AU777860B2 - Nitroreductase enzymes - Google Patents

Nitroreductase enzymes Download PDF

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AU777860B2
AU777860B2 AU24511/00A AU2451100A AU777860B2 AU 777860 B2 AU777860 B2 AU 777860B2 AU 24511/00 A AU24511/00 A AU 24511/00A AU 2451100 A AU2451100 A AU 2451100A AU 777860 B2 AU777860 B2 AU 777860B2
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nitroreductase
gin
ala
val
pro
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Gill Anlezark
Nigel Minton
Thomas Vaughan
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Health Protection Agency
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Description

WO 00/47725 PCT/GB00/00431 -1- NITROREDUCTASE
ENZYMES
The present invention relates to polypeptides and proteins .having nitroreductase activity, to DNA and genes encoding these nitroreductases and to methods of obtaining such enzymes, DNA and genes.
A number of cancer therapies are based upon or exploit the conversion of a non-toxic prodrug into a toxic derivative.
One example concerns the monofunctional alkylating agent CB1 954, which exhibits extreme toxicity towards the Walker 256 rat carcinoma as a result of the presence of a DT-diaphorase enzyme (DTD) which reduces the 4nitro group of CB1954 to give a highly cytotoxic 4-hydroxylamine (4HX) derivative. CB1 954 does not have the same effect on human carcinomas because human cells lack this enzyme but would be effective against human tumours if an enzyme such as DTD were externally supplied, e.g.
in a Directed-Enzyme Prodrug Therapy (DEPT). The rat DTD, however, has a relatively poor specific activity for CB1954. The E.coli B nitroreductase enzyme (NfnB) was isolated as a more effective alternative and is the subject of EP-A-0540263. It exhibits a higher specific activity for CB1 954, compared with the rat enzyme and is, therefore, currently the preferred enzyme in anti-cancer DEPT strategies.
Whilst the known E.coli enzyme receives widespread attention from cancer biologists seeking to develop gene based DEPT strategies, it has a number of drawbacks. These mostly relate to its activity against the preferred prodrug, CB1954 it has a relatively high Km and low Kcat, and converts CB1954 into equimolar amounts of a relatively innocuous 2-hydroxylamino derivative (2HX) in addition to the highly cytotoxic 4-hydroxylamino species (4HX).
In relation to this specific prodrug, it is hence desired to provide an 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 062837999 N0.525 D005 -2alternative to the known E.coli enzyme.
Additionally, and more generally, analogues of CB1954 and prodrugs other than CB1954 are known and further such precursors of potential toxic agents may become the focus of future therapies. In relation to all of these it is desired to provide further enzymes capable of use in converting prodrugs into drugs, e.g. for clinical uses.
It would be advantageous if at least preferred embodiments of the Invention were to provide nitroreductase enzymes, in particular nitroreductase enzymes for converting CB1954 and analogues thereof into drugs. It would be advantageous if at least preferred embodiments of the invention were to provide DNA and genes encoding nitroreductases, which DNA and genes in particular are incorporated into pharmaceutical compositions for prodrug therapies.
The present invention is based upon the discovery, purification, gene sequencing and/or expression of nitroreductases in bacteria and other microorganisms with hitherto unknown properties in converting prodrugs such as CB1954 into toxic derivatives.
These nitroreductases possess properties which alone or in combination offer potential improvements compared with the known enzymes In this technology. The 20 nitroreductases of the invention may be divided into different families based upon such characteristics as activity, product spectrum and/or amino acid sequence, and each given nitroreductase may fall into more than one of these families.
The present invention provides, in a first aspect, a nitroreductase characterised in that it preferentially reduces CB1954 to a cytotoxic 4-hydroxylamine (4HX) derivative instead of a non-cytotoxic 2-hydroxylamine derivative, wherein the nitroreductase has at least 70% identity with a sequence selected from the group consisting of SEQ ID Nos: 2, 4, 10, 12, 17, 20, 21, 23, 25 and 29.
30 The enzymes of this aspect of the present invention confer the advantage that the product they generate from CB1954 contains a greater proportion of the cytotoxic 4HX
S*
COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 062837999 N0.525 0006 3 derivative than the non-cytotoxic 2-hydroxylamino derivative. In preferred embodiments of the invention, the product is substantially entirely the cytotoxic derivative. The enzymes may hence be more efficient that those of the art as the enzymes of the invention produce more cytotoxic product for a given amount of prodrug.
The present invention further provides, in a second aspect, a nitroreductase enzyme, characterised in that it reduces a prodrug to a toxic derivative with a Km of less 700 micromolar, wherein the prodrug is selected from CB1954 and analogues thereof or other bioreductive drugs (Denny et al, B.J. Cancer, 1996, 74, pp S32-S38) and wherein the nitroreductase has at least 70% identity with a sequence selected from the group consisting of SEQ ID Nos: 2, 12, 17, 20, 21 and 23. The enzymes of the second aspect of the invention offer an advantage over the known E.coli-derived enzyme in that they have a lower K, of E.coli NfnB for CB1954 is around 862 micromolar) and thus have a higher affinity for substrate. Twenty nitrogen mustard analogues of CB1954 are described by Friedlos et aJ (J Med Chem, 1997, 40, 1270-1275).
More preferably, the K, of the enzymes of the second aspect of the invention is less than 300 micromolar.
In a third aspect, the present invention provides a nitroreductase characterised in that it reduces a prodrug to a toxic derivative with a Kt of at least 8, wherein the nitroreductase has at least 70% identity with a sequence selected from the group consisting of SEQ ID Nos: 10, 12, 17, 20, 21 and 23. In one embodiment, the present invention provides a nitroreductase enzyme characterised in that It reduces a prodrug to a toxic derivative with a Kca of at least 8, wherein the prodrug is selected from CB1954 and analogues thereof.
The enzymes of this aspect of the invention offer an improvement over that of the art, 30 specifically the E.coli enzyme, in that they have an improved Kc i.e. a higher value than for Ecoli NfnB Indicating a higher turnover of substrate by the enzyme. In preferred embodiments of this aspect of the invention, the K a of the enzymes is at least In a fourth aspect of the invention, there is provided a nitroreductase characterised in COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 062837999 NO.525 0007 4 that it reduces CB1954 to a toxic derivative, it reduces SN23862 to a toxic derivative, it can use both NADH and NADPH as electron donor and in that it shares no more than sequence identity with the Ecoli NfnB sequence, and wherein the nitroreductase has at least 70% identity with a sequence selected from the group consisting of SEQ ID Nos: 10, 12, 17 and 29. Preferably, the sequence identity is about 25% or less, this sequence identity being measured using the MEGALIGN (registered trade mark) software.
It has already been discussed how the known E.col nitroreductase is well characterised and is fully sequenced. The nitroreductases of the fourth aspect thus represent a class of enzymes having nitroreductase activity, or being nitroreductaselike, which nevertheless are so different in amino acid sequence from the E.coli enzyme as to represent a separate family of nitroreductases.
This aspect of the invention thus advantageously provides a further class of nitroreductase enzymes for use e.g. in prodrug therapies.
The invention still further provides, in a fifth aspect, a nitroreductase enzyme characterised in that it reduces CB1954 or an analogue thereof to a toxic derivative, in 20 that it shares at least 50% sequence identity with the rat DTD sequence and in that it Sdoes not contain a domain that is the same as or corresponds to amino acids 51 to 82 *of the rat DTD sequence.
Sequence identity is suitably measured in the same way as described above in relation to the fourth aspect.
To determine whether a given nitroreductase contains a domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence, the amino acid sequence of the given nitroreductase and of the rat DTD sequence are aligned using a S 30 conventional sequence alignment program, such as MEGALIGN (registered trade *i eO0 Oo •oo• COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 062837999 N0.525 008 mark) made by DNASTAR, Inc.
If the alignment program indicates that there are no amino acids in the given sequence that, following the algorhythm of the program, are held to correspond to those at positions 51-82 of the rat DTD sequence then it is concluded that the rat domain is lacking from the given sequence.
This aspect of the invention thus provides a further class of nitroreductase enzymes for conversion e.g. of prodrugs into drugs. A nitroreductase in this class may also be obtained by deleting amino acid residues that correspond to residues 51-82 of the rat DTD from a known mammalian enzyme.
The nitroreductases of the Invention may also be NADPH dependant. This property further distinguishes some aspects of the invention from the known E.coi enzyme and the rat DTD.
It has been found that enzymes having one or more of the properties described may be obtained from bacteria of the family Bacillus, in particular a Bacillus selected from B.
amyloliquefeciens, B. subtilis, B. pumilis, B. lautus, B. themoflavus, B. licheniformis S 20 and B. alkophilus. This finding is of surprise in that at least three nitroreductase enzymes have been found in some species, in particular B. subtilis, B. lautus and B.
pumilis, and as nitroreductases having the advantageous properties of the invention have not hitherto been identified in these bacteria, the currently used nitroreductase being obtained from E.coli.
As described herein, a nitroreductase has a sequence selected from SEQ ID Nos 2, 4.
6, 8, 10, 12, 14, 16, 17, 18, 19, 20, 21, 23, 25, 27 and 29.
*I It has further been found that nitroreductases according to the invention may fall into 30 more than one aspects of the invention. It is hence preferred that a nitroreductase of the invention possesses the properties of at least two aspects of the invention, and more preferably at least three aspects of the invention.
@Io COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HA#CK +61 2 99255911 4 062637999 NO.525 P009 Aspecific embodiment of the Invention is a nitroreductase of SEQ ID NO:2 obtained fro B.ayoiufcesti enzyme Converts CD1 94 into substantially Only the c~tto~c eriatvehene allngint th frstasectofthe invention, but also has a Km that Is improved compared to the E-coll enzyme, hence failing also into the second aspect of the invention.
A further specific embodiment of the invention is a nitroreductase from B. Subtiis,
SEQ
ID NO:9. This enzyme has a better than the E-colI enzyme, iUs being about compared with about 6 for the E.coii enzyme, and hence falls into the third aspect of the invention. Additionally, tWs. enzyme falls into the fourth aspect of the invention in that it reduces both CB1954 and SN23862 but shares less than 30% sequence identity with the Ecoll sequence. Another B. subtilis enzyme, SEQ ID NO:11 Is similarly in both the third and fourth aspects of the invention, having a Kct of about From the examples set out below it will be apparent how the further specific embodiments of the invention fall into at least two and even three aspects of the Invention.
The enzymes of the invention are of use in enzyme direclted prodrug therapy.
20 Accordingly, it is preferred that they are provided in purified formn.
A sixth aspect of the invention provides a pharmaceutical composition comprising a nitroreductase enzyme according to any of the first to fifth aspects of the invention in combination with a pharmraceuticaliy acceptable carrier.
25 In a seventh aspect, the invention provides use of a nitroreductase having at least identity with a sequence selected from SEQ 10 Nos: 2, 4, 10, 12, 17, 20, 21, 23, and 29, for the manufacture of a medicament for prodrug therapy.
30 As mentioned above, the nltroreductase of the Invention are of use in COMS IDNo: SBMI-00906201 Received by I13 Australia: Time 15:23 Date 2004-09-09 WO 00/47725 PCT/GB00/00431 -7therapies such as directed-enzyme prodrug therapies. In these therapies, it is required to deliver the nitroreductase to the target site. This delivery can be achieved by delivering the enzyme itself or by delivering a DNA or gene coding for the enzyme.
In an example of the enzyme of the invention in use, a pharmaceutical composition is designed for a directed-enzyme prodrug therapy, and comprises a pharmaceutically acceptable carrier and a compound for converting a prodrug into a drug, wherein a compound is composed of at least a nitroreductase according to any of the first to fifth aspects of the invention conjugated to a targeting moiety.
The targeting moiety can suitably comprise an antibody specific for a target cell. Alternatively, the targeting moiety is a moiety preferentially accumulated by or taken up by a target cell.
A further example of delivery of the enzyme of the invention is achieved in a gene therapy-based approach for targeting cancer cells, as described in WO 95/12678. As described by Knox R.J. et al, the basis of this further prodrug therapy is delivery of a drug susceptibility gene into target, usually tumour or cancer, cells. The gene encodes a nitroreductase that catalyses the conversion of a prodrug into a cytotoxic derivative. The nitroreductase itself is not toxic and cytotoxicity used to treat the tumour cells arises after administration of a prodrug which is converted into the cytotoxic form. A bystander effect may be observed as cytotoxic drug may diffuse into neighbouring cells.
Thus, in this gene-based therapy, the nitroreductase is expressed inside a cell, in contrast to other delivery systems in which, for example, the enzyme itself is delivered accompanied by a targeting moiety.
Targeting of gene-based therapies may be achieved by providing a virus or WO 00/47725 PCT/GB00/00431 -8liposome with altered surface components so that the delivery vehicle is recognised by target cells. Typically, transcriptional elements are chosen so that the gene coding for the nitroreductase enzyme will be expressed in the target cells, and preferably substantially only in the target cells. A number of viral-based vectors are suitable for this delivery. Retro-viral based vectors typically infect replicating cells. Adenoviral vectors and lentiviral-vectors are also believed to be suitable.
This delivery technology has been demonstrated by Bridgewater et al (Eur J Cancer 31a, 236-2370, 1995). A recombinant retrovirus encoding a nitroreductase was used to infect mammalian cells, it being observed that infected cells expressing the nitroreductase were killed by application of CB1954.
Accordingly, a further aspect of the invention provides the use of a DNA sequence coding for a nitroreductase of the invention in manufacture of a medicament for prodrug therapy.
The medicament may take the form of a viral vector, comprising a DNA encoding the nitroreductase of the invention operatively coupled to a promoter for expression of the DNA. The medicament may take the form of a mini-gene comprising a DNA operatively linked to a promoter for expression of the DNA, the mini-gene being suitable for inclusion or incorporation into a targeting vehicle such as a microparticle.
Thus, an embodiment of the invention provides a viral vector comprising a nucleotide sequence encoding a nitroreductase according to any of aspects 1 to 5 of the invention, which nitroreductase converts a prodrug into a cytotoxic drug, and also a kit comprising the viral vector and the prodrug, and also a method of treatment of tumours which comprises administering an effective amount of the viral vector together with an effective amount of the prodrug.
WO 00/47725 PCT/GBOO/00431 -9- The preparation and administration of these viral vectors may be substantially as described in WO 95/12678, the contents of which is incorporated herein by reference. The present invention relates to providing nitroreductase enzymes and genes and DNA coding therefore.
The uses of those enzymes and genes may be as set out in WO 95/12678.
A nitroreductase can also be delivered by putting a gene of the invention into a bacteria that selectively colonises tumours, such as a clostridial (Lemmon et al, Gene Therapy, 1997, 4, 791-796) or Salmonella species.
A further aspect of the invention provides an isolated DNA encoding a nitroreductase according to any of the first to fifth aspects of the invention.
The DNAs of this further aspect of the invention, and also the DNAs incorporated into vectors of the invention, preferably comprise a sequence which is selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 22, 24, 26 or 28, together with fragments, derivatives and analogs thereof retaining nitroreductase activity according to one of the first to fifth aspects of the invention. The fragments, derivatives and analogs are suitably selected from sequences which retain at least 70% identity with the specific embodiments of the invention, or preferably at least 90% identity and most preferably at least 95% identity.
The enzymes of the invention can also be obtained by purification from cell extracts and may also be obtained by recombinant expression of DNA. A still further aspect of the invention lies in a method of preparing a nitroreductase enzyme, comprising expressing a gene in a bacterial cell, wherein the gene codes for a nitroreductase enzyme of the invention.
In an example of the invention described below in more detail, the gene expressed is a Bacillus gene or is a gene obtained by substitution, deletion and/or addition of nucleotides in or to a Bacillus gene.
WO 00/47725 PCT/GB00/00431 The invention also provides the use of a nitroreductase according to any of the aspects of the invention in manufacture of a medicament for antitumour therapy, and the use of a compound comprising a nitroreductase according to any aspect of the invention conjugated to a targeting moiety in manufacture of a medicament for anti-tumour therapy.
The invention is now illustrated by the following specific examples and in the accompanying sequence listing in which: SEQ ID NO: 2 is a nitroreductase from B.amyloliquefaciens (coded for by SEQ ID NO: 1) and designated "Bam YrwO"; SEQ ID NO: 4 is a nitroreductase from B.subtilis (coded for by SEQ ID NO: 3) and designated "Bs YwrO"; SEQ ID NO: 6 is a nitroreductase from B.subtilis (coded for by SEQ ID NO: 5) and designated "YrkL"; SEQ ID NO: 8 is a nitroreductase from B.subtilis (coded for by SEQ ID NO: 7) and designated "YdeQ"; SEQ ID NO: 10 is a nitroreductase from B.subtilis (coded for by SEQ ID NO: 9) and designated "Ydgl"; SEQ ID NO: 12 is a nitroreductase from B.subtilis (coded for by SEQ ID NO: 11) and designated "YodC"; SEQ ID NO: 14 is a nitroreductase from E.coli (coded for by SEQ ID NO: 13) and designated "YabF" SEQ ID NO: 16 is a nitroreductase from E.coli (coded for by SEQ ID NO: 15) and designated "YheR"; SEQ ID NO: 17 is a nitroreductase from H.influenzae; SEQ ID NO: 18 is a nitroreductase from T.aquaticus; SEQ ID NO: 19 is a nitroreductase from Synechocystis sp PCC 6803; SEQ ID NO: 20 is a nitroreductase from A.fulgidus; SEQ ID NO: 21 is a nitroreductase from A.fulgidus.
SEQ ID NO: 23 is a nitroreductase from Campylobacterjejuni (coded for by SEQ ID NO: 22); SEQ ID NO: 25 is a nitroreductase from Porphyromonas gingivalis WO 00/47725 PCT/GBOO/00431 -11- (coded for by SEQ ID NO: 24); SEQ ID NO: 27 is a nitroreductase from Yersinia pestis (coded for by SEQ ID NO: 26); and SEQ ID NO: 29 is a nitroreductase from Helicobacter pylori (coded for by SEQ ID NO: 28).
The invention is also illustrated by reference to the accompanying Tables 1-4 and Figures 1 and 2, in which Figs 1 and 2 show sequence comparisons as set out in more detail in Example 8.
Example 1 A Nitroreductase Enzyme/Gene from Bacillus amyloliquefaciens Briefly, extracts of Bacillus amyloliquefaciens were shown to possess nitroreductase activity. To purify this activity, crude cell extracts were subjected to ammonium sulphate, fractionation and anion exchange chromatography. The purified material was subject to N-terminal amino acid sequence analysis and the information obtained used to cloned the gene via a PCR-based strategy. Following determination of its nucleotide sequence the gene was overexpressed in E. coli and the resultant recombinant protein purified and characterised see table 1.
This analysis showed that the enzyme had properties which were distinct from that of E.coli NfnB. Thus the protein had a more favourable Km for CB1954 (1.5-fold lower than the E. coli B NfnB) and furthermore converted CB1954 into the 4HX form alone. It also differed from the E. coli B NfnB in that the enzyme showed no activity against the prodrug SN23862.
The isolated enzyme/gene represents a significant improvement over the E.co/iNfnB enzyme with respect to its activity against the prodrug CB1 954 ie., it produces only the 4HX derivative and has an improved Km for CB1954.
WO 00/47725 PCT/GBOO/00431 12- A comparison of the amino acid sequence of the isolated enzyme revealed that it shared a very low level of homology to the rat DTD but exhibited high homology (70% sequence identity) with the predicted product of a gene that has been discovered in the Bacillus subtilis genome sequencing project, designated ywrO. On this basis, we have designated the cloned Bacillus amyloliquefaciens gene ywrO, and its encoded enzyme YwrO.
YwrO BAM is a tetrameric flavoprotein (monomeric molecular mass approximately 22.5 kDa by SDS-PAGE, native molecular mass approximately 90 kDa by gel filtration). Although it shares sequence homology with rat DTD it differs in its enzymic properties in that it can use only NADPH as cofactor (Km 40 pM). In common with DTD it can reduce CB1954 but not SN23862, reduction of CB1954 resulting in formation of the 4HX product only (Km 617 pM, kca, It shows a high affinity for the quinone menadione (Km 3.4 pM) and has azoreductase and flavin reductase activity (Km for FMN 53 uM, Km for FAD 209 pM).
In more detail, N-terminal amino acid sequencing of the purified Bacillus amyloliquefaciens nitroreductase enzyme resulted in the following sequence,Met-Lys-Val-Leu-VaI-Leu-Ala-Val-His-Pro-Asp-Met-Glu-Asn-Ser- Ala-Val-Asn. When this sequence was used to search available protein databases strong homology was noted with the predicted amino acid sequence of a hypothetical protein, YrkL, identified in the Bacillus subtilis genome sequencing project. Significant homology was also evident with two proteins, YabF and YheR, identified during the course of the determination of the Escherichia coli genome. These three hypothetical proteins shared weak homology with a number of mammalian quinone reductases and NAD(P)H-oxidoreductases, such as the rat DTD.
In view of this observation, a strategy was formulated whereby sequence homology between the identified bacterial proteins, together with the WO 00/47725 PCT/GBOO/00431 13determined N-terminal amino acid sequence of the discovered Bacillus amyloliquefaciens enzyme, was used to amplify a region of the desired encoding gene from the Bacillus amyloliquefaciens genome. The one primer utilised in PCR was a degenerate oligonucleotide sequence which corresponded to a DNA sequence capable of coding for the N-terminal octa-peptide Val-His-Pro-Asp-Met-Glu-Asn. It was composed of the following nucleotides, 5'-GTNCAYCCNGATATGGARAA-3', where Y indicates the presence of a T or C, R indicates the presence of A or G, and N indicates the presence of either T, C, G or A. The second primer was based on the hypothetical sequence His-Gly-Trp-Ala-Tyr-Gly which was found to be entirely conserved between the hypothetical bacterial proteins YrkL (Bacillus subtilis) and YabF (E.coli), and partially conserved in YheR coll). The degenerate oligonucleotide mixture synthesised corresponded to the antisense DNA coding strand, viz., 5'-CCRTANGCCCANCCRTG-3'.
E.coli YheR (90-95) Arg Gly Phe Ala Ser Gly E.coli YabF (84-89) His Gly Trp Ala Tyr Gly B.subtilis YrkL (85-90) His Gly Trp Ala Tyr Gly The two primers were employed in PCR using chromosomal DNA isolated from Bacillus amyloliquefaciens and an amplified DNA fragment of the expected size (approximately 230 bp) obtained. This was cloned into plasmid pCR2.1TOPO (Invitrogen) and its nucleotide sequence determined.
Translation of the sequence obtained demonstrated the presence of an open reading frame which encoded a polypeptide which shared 66% sequence similarity with YrkL.
To obtain the entire structural gene, an approach was employed based on inverse PCR. In essence, B. amyloliquefaciens DNA was cleaved with the restriction enzyme Styl and the fragments generated circularised through their subsequent incubation with DNA ligase. The ligated DNA was then used as the template for a PCR employing two divergent primers based on WO 00/47725 PCT/GBOO0/0043 1 14the sequenced 220 bp fragment. These were BamNTR11 GCTTATTGACCGCTGAG-3') and BamNTR14 A 2.9 kb fragment was generated, cloned into pCR2.1TOPO (Invitrogen) and the sequence of the insert determined. This allowed the identification of the nucleotide sequence of the remaining parts of the B.
amyloliquefaciens gene. Using this information, a contiguous copy of the entire structural gene was amplified from the B. amyloliquefaciens chromosome using primers which encompassed the translational start codon (5'-GGTGTGATACATATGAAAGTATTG-3') and resided 3' to the translational stop codon (5'-CGGGGATTCGAATTCTTTCTCAGG-3'). The primer at the 5'-end of the gene was designed such the sequence immediately 5' to the ATG start codon became CAT. This change created an Ndel restriction site (CATATG), thereby allowing the cloning of the gene into the equivalent site of the expression vector pMTL1015. This manipulation facilitated the subsequent overexpression of the gene, as insertion of the gene at this point positions the start codon at an optimum distance from the vector borne ribosome binding site.
The strategy employed to clone the BM YwrO gene could be similarly employed to clone further genes encoding novel nitroreductases. This would involve purifying the desired enzyme activity from a cell lysate, and then determining the N-terminal sequence. The data obtained could then be used to design an oligonucleotide primer corresponding to the sense strand of the DNA encoding part or all of the determined amino acid sequence. This primer could then be used, in conjunction with a second primer, to amplify part of the gene encoding the nitroreductase from the chromosome of the bacterial host using PCR. The second primer would correspond to the antisense strand of an internal portion of the targeted gene. Its design would be based on regions of homology which are conserved amongst the type of nitroreductase family that is sought. Thus, in the case of the DTD-like family, the oligonucleotide would, for example be based on the conserved motif His-Gly-Trp-Ala-Tyr-Gly amino acid WO 00/47725 PCT/GBOO/0043 1 residues 85-90 in the BS YrkL protein). In the case of the NfnB-like family, the oligonucleotdie could be based on the motif Glu-Arg-Tyr-Val-Pro-Val- Met amino acid residues 170-176 in the BS YodC protein).
Such amplified fragments could then be cloned and sequenced, and new primers designed based on this sequence to isolate the flanking regions of the gene by PCR. Once these have been cloned and sequenced, the entire, contiguous structural gene may be amplified using primers which extend beyond the 5' and 3' end of the translational start and stop codons.
Cloning of genes encoding novel nitroreductases may also be achieved without recourse to N-terminal sequencing of the enzyme, or even its purification. This would involve basing the sequence of both of the oligonucleotides used in the initial PCR reaction on amino acid sequence motifs conserved amongst the two identified nitroreductase families.
Thus, in the case of the NfnB-like family, a sense primer ATTTCTAAAGAAGAGCTGACGGAA-3') based on the motif Ile-Ser-Lys-Glu- Glu-Leul-Thr-Glu amino acid residues 13 to 20 of BS YodC) could be employed with the an antisense primer CATTACCGGTACATAGCGTTC-3') based on the sequence motif Glu-Arg- Tyr-Val-Pro-Val-Met amino acid residues 170 to 176). In the case of the DTD-family a sense primer 5'-CATCCGGATATGGAAAAT-3') based on the motif His-Pro-Asp-Met-Glu-Asn amino acid residues to 9 to 14 of BM YwrO) could be employed with the an antisense primer (eg., 5'-TCCATATGCCCATCCATA-3') based on the sequence motif Tyr-Gly- Trp-Ala-Tyr-Gly amino acid residues 85 to 90). Once amplified, the rest of the gene could be isolated using the same procedure as outlined above.
Example 2 Bacillus subtilis Nitroreductases WO 00/47725 PCT/GBOO/00431 -16- As indicated above in Example 1, comparative analysis of the B. subtilis genome sequence with the amino acid sequence of the isolated B.
amyloliquefaciens enzyme demonstrated the existence of an enzyme (YwrO) which shared 70% sequence identity. Unexpectedly, B.subtilis was found to possess two homologues, YrkL and YdeQ, which share 54% and 51% sequence homology, respectively, with the B. amyloliquefaciens enzyme. All three enzymes share no homology with the E.co/i B NfnB.
They do, however, exhibit weak similarity 25%) to the rat DT- Diaphorase (DTD). Whilst these proteins share a low level of sequence similarity to DTD, and other mammalian equivalents, they are characteristically smaller. This is because of the absence of an extensive internal protein domain at the N-terminus of the protein. Thus, the functional equivalent domain of the rat DTD between amino acid residues 51 to 82, are missing from the BM YwrO protein. In addition, the rat DTD has an extra COOH-terminal domain. These bacterial enzymes are thus distinct from their mammalian equivalents.
A further analysis of the B.subtilis genome, demonstrated that two homologues of the E.coliNfnB gene were present. Their encoded proteins (Ydgl and YodC) share a barely detectable level of sequence conservation with EC NfnB, of around 20% sequence identity.
Bacillus subtilis was thus found to carry at least 5 different enzymes with nitroreductase activity. These are split into two families, thus;- DTD-like 3 members:- YwrO, YrkL, YdeQ NfnB-like 2 members:- Ydgl, YodC Example 3 Recombinant Production of Nitroreductases from Bacillus subtilis The DNA encoding all 5 B.subtilis nitroreductase enzymes were cloned WO 00/47725 PCT/GBOO/00431 17from genomic DNA using PCR and the resultant genes, following authentification by nucleotide sequencing, subcloned into a propriety CAMR expression vector (pMTL1015). The expression clones generated have been used to overproduce each of the 5 proteins and the enzymic activity of each assessed in crude lysates. This analysis has demonstrated that whilst the B.subtilis YwrO shares similar properties to the B.
amyloliquefaciens homologue converts CB1954 to the 4HX derivative alone, but is inactive against SN23862), YrkL and YdeQ have no activity against either of the two prodrugs tested (CB1 954 or SN23862) but they may be active against other prodrugs.
Despite the extremely limited sequence similarity to EC NfnB, Ydgl and YodC are active against both CB1 954 and SN23862. They do, however, produce both the 2HX and 4HX derivatives of CB1954. Their characterisation has shown that they turn over CB1 954 at higher rates than EC NfnB (YodC kcat 58, Ydgl ka, 30.3 cf 6 for NfnB). Both show a high affinity for menadione and flavins, but they differ in that whereas Ydgl uses both NADH and NADPH, YodC shows a preference for the latter. The native molecular mass of YodC (approximately 90kDa) indicates that it is tetrameric (molecular mass estimated from amino acid sequence and by SDS-PAGE being approximately 22 kDa) whereas Ydgl appears to be a dimer in the native state (molecular mass by gel filtration approximately 49 kDa).
These finding are further illustrated in Table 2.
Example 4 Bacillus lautus Bacillus pumilis nitroreductases From 103 soil sample isolates tested, two strains (Bacillus pumilis CP044 and Bacillus lautus CP060) had been previously chosen as possessing extracts which showed the most rapid reduction of both CB1 954 and WO 00/47725 PCT/GBOO/00431 -18- SN23862. Purification experiments demonstrated that the activity in both extracts was distributed across three distinct peaks. The presence of more than one enzyme activity is consistent with our discovery of multiple forms of proteins in Bacillus able to turnover prodrugs. Eventual purification of the three enzymes of B. pumilis CP044 revealed that no one candidate exhibited properties which were an improvement on the E.coli NfnB enzyme. In contrast, the proteins in peak 1 and peak 3 of the B.lautus CP060 were determined to offer advantage over NfnB.
Thus, whilst the enzyme in peak 1 did not produce the required 4HX derivative of CB1954, it exhibited a 4-fold lower Km with the prodrug SN23862. The enzyme of peak 3 was, however, deemed to be of greatest value as it converted CB1954 solely into the 4HX derivative and had a Km approximately 4-fold lower than NfnB. Furthermore, it also had activity against SN23862. In this respect it shares the properties of both the Bacillus DTD-like family it produces only the 4HX derivative) and the NfnB-like family it is active against SN23862) these findings are illustrated in Table 3.
Example N-terminal Sequencing of B. lautus Nitroreductase Electrophoretic separation of the peak 3 demonstrated that 4 protein bands were present which could account for the observed prodrug activity. All four were subjected to N-terminal amino acid sequencing and the activity localised to the fourth protein band from which the nitroreductase may be purified.
Example 6 Detection of Nitroreductase Activity in Thermophile Extracts As an alternative source novel enzymes, a preliminary screen of CAMRs WO 00/47725 PCT/GB00/00431 -19thermophile collection was undertaken. Enzymes from this source may have the advantage of greater stability, and therefore longevity of action.
Strains were selected on the basis either of sensitivity to CB1 954, or those which are resistant but which impart a yellow/golden coloration to agar containing prodrug.
Two of these strains thermoflavus and B. licheniformis) generated the cytotoxic 4HX form and were selected for further study.
Example 7 Identification Of Further Nitroreductase Enzymes Having identified the two families of nitroreductase in Bacillus, a search was undertaken of both finished and unfinished genomes for homologues, using YwrO and YodC/NfnB. On the basis of this search homologues of YwrO were identified in the genomes of Yersiniapestis and Porphyromonas gingivalis, and homologues of NfnB in the genomes of Pyrococcus furiosus, Haemophilus influenza, Synechocystis PCC 6803, Campylobacter jejuni, Archaeglobus, Helicobacter pylori, Heliocbacter fulgidus and Thermus aquaticus.
In addition to the above, two E.coligenes were found to be homologues of rat DTD and YwrO, and were designated Yher and YabF. They were discovered to share the characteristic of YwrO in that they lack the internal protein domain found in the rat DTD enzyme and functional mammalian homologues.
P.gingivalis YwrO homologue P.gingivalis YwrO homologue is a dimeric flavoprotein with native molecular mass estimated by gel filtration at 40 kDa. Although it shares sequence homology with DTD and forms only the 4HX reduction product of CB1 954 WO 00/47725 PCT/GBOO/00431 (Km 1200/pM, kca, it differs from DTD in that it is active with SN23862 and it can only use NADH as cofactor (cf DTD which can use either NADH or NADPH and is inactive with SN23862). It can reduce azodyes but it is inactive with menadione or flavins.
(ii) C.jejuni NfnB homologue C.jejuni NfnB homologue produces only the 4HX reduction product of CB1954 (Km 143 kcat 11.2) using NADPH as cofactor and it is also active with SN23862. It can use the quinone menadione as substrate as well as azodyes and the flavins FMN and FAD.
(iii) Archaeoglobus fulgidus NfnB homologue Archaeoglobus fulgidus NfnB homologue is a dimeric flavoprotein of 42 kDa native molecular mass, producing the 4HX derivative of CB1954 only (Km 690 jM, kcat 56.2) using NADPH as cofactor. It is also active with SN23862 and menadione (Km 9pM), but does not decolourise azodyes and has only weak flavin reductase activity.
(iv) H.influenzae and H.pylori NfnB homologues Both these enzymes are dimeric flavoproteins and form the 4HX reduction product of CB1954 using NADPH in preference to NADH, but have no activity with azodyes. The former also lacks activity with the quinone menadione and flavins FMN or FAD. Both however have weak activity with SN23862 and may be active with other prodrugs.
Y.pestis nfnB homologue and Synechocystis YwrO homologue Both these proteins reduce CB1954 but produce only the relatively nontoxic 2HX derivative using NADPH as cofactor. They do however show WO 00/47725 PCT/GBOO/00431 -21 activity with SN23862 and the former can also reduce azodyes.
Example 8 Comparison of Nitroreductase Sequences We compared the amino acid sequences of nitoreductases according to the invention with each other and with known rat, human and E.coli sequences, and the results are illustrated in Figures 1 and 2. In Figure 1, rat, mouse and two human sequences make up the first four lanes for comparison purposes. It is evident that nitroreductases of the invention are lacking a sequence from positions 51-82 of the rat sequence.
In Figure 2, sequences of nitroreductases of the invention are compared with the known E.coli sequence, which is designated nfmB in the secondto-last lane.
The invention thus provides nitroreductase enzymes, DNA and genes therefor and methods of obtaining such enzymes and of using the enzymes and DNA coding therefor in clinical applications.
SUBSTITUTE SHEET (RULE 26) 22 ENZYME M.Wt CB1954 SN23862 (Kda) Product Km kcat Km keat .E-coll N&nB 24 2/4HX 862 6.0 2500 26.4 Rat DTD 33 4HX 826 0.07 inactive inactive Bani YwrO 22 4HX 617 2.0 inactive inactive Table 1: Characteristics of nitroreductase enzymes from Bacillus amyloliquefaciens NfnB nitroreductase of E. coli 13; DTD DT Diaphorase;, and; Barn YwrO cloned Bacillus amyloliquefraciens nitrareductase a a a a a DTD-like Family NfnB-like Family YwrO YrkL YdeQ YdgI YodC Homology 70% 54% 51% 25% 24% CB1954 41{X inactive inactive 2/4HX 2/4HX SN23862 inactive inactive inactive active active Table 2: Characteristics of nitroreductase enzymes found in the Bacillus subtilis genome *DTD-like family homologies are to the Bacillus amyloliquefaciens YwrO, NfnB-like family homologies are to the' E.coli B nitroreductase.
23 I i
ENZYME
ACTIVITY
M.Wt (kDa) ENZYMEM.WtCB 1954 SN23862 Kzm ACIIT (ka Prdc IKI B. pumilis CP044 Peak I Peak 2 Peak 3 4HX 4HX 2/4H-X V. low >1000 999 1* 4 1 B.lautus CP060 Peak 1 Peak 2 Peak 3
ND
ND
ND
325 none active 2HX 4HX 4HXK 21] >2000 1 L 257 Table 3: Fractionation of nitroreductase activity in cell extracts of Bacillus taut us and Bacilluspumilis STRAIN CB1954 SN23862 Product NADH NADPH NADH NADPH 1078 2/4HX 13.8 22.6 8.5 17.6 21222 2/13HX 36.6 56.0 33.4 62.8 6012 b 4>2HXK 15.2 37.8 8.2 35.2 6013 c 2HX 9.8 49.4 6.4 39.0 6031 d 21fX 11.9 42.1 8.2 33.8 6036 2HX 10.7 26.7 7.3 26.2 6044 2HX 4.0 21.3 4.5 9.9 Table 4: Characteristics of nitroreductase activity of thermophiles identified as being sensitive to CP1954 [Identified as Bacillus thermoflavusa, Bacillus lichenformisb Bacillus licheniformis', Bacillus alkophilus
I
23a In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.
It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.
go 0 00 oooo EDITORIAL NOTE APPLICATION NUMBER 24511/00 The following Sequence Listing pages 1 to 32 are part of the description. The claims pages follow on pages 24 to 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 062837999 NO.525 001? SEQUENCE LISTING <110> Health Protection Agency.
<120> Nitroredactase Enzymes <130> MRM/CMS/2122 8 <2.50> 3B .9903019.9 <151> 1999-02-10 <160> 29 <170> Patentin version 3.1 5
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0 0 0900 0 <210> 2.
<211> 525 <212> DNA <213> Thacillus amyloliquefaciens <220> <221> CDS <222> .(525) <223> <400> 1 gtg aaa gta Met Lye Val 1 ttg gta tea gcg gtt Cac Leu Val Leu Ala Val Iia 5 gac atg gag Sac Asp Met Glu Agn tea gcg Ser Ala gte aat aag gca Val An Lye Ala egg gee gaa gas Trp Ala Glu Glu tta Leu 25 &aa aaa cat gat Lye Lys His Asp gaa etc acg Glu Leu Thr COMS ID No: SBMI-00900201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092804 15:01 GRIFFITH H-ACK< +61 2 99255911 4 062837999NO55Di NO. 525 P018 gtc Cgt gag Val Arg Gin aag gaa Cgt LYS Gin Arg, cct tat aaa gaa tat CCU gac ggg caa atc gat gcg gaa Len Tyr Lys Glu Tyr Pro Asp Sly Gin Ile Asp.Ala Gin 40 4 cag ctg tgt gaa cag tat gac cgg etc gte ttt caa .ttt GJn Lou Cys Gin Ginl Tyr ASP Arg Ile V'ai Phe Gin Phe 55 Ccg ctg tat tgg tac agt gcg cct cog. ott.
Pro Len Tyr Tx-p Tyr Ser Ala Pro Prd Leu .70 cat gtg Ctg tcg tacgg9c His Va). Len Ser Tyr Sly tgg goc tac Trp Ala Tyr 9gc Gly.
tt& aa Lett Lys too aaa Ser Lys* ac-a tgg &tg gat Thr TrpMeAs So Sly Lys Ala Len 144.
192 240.
28 336 3.84 oat grgc aaa His Sly Lys gca tac cag.
Al-iaTyr Gin ttg atg Ctg Ieu Met Lou get gtt tcc gta Ala Val Ser*Val aec ceo att ace Aen His Ph. Thr 120 ggt gcc ggA gag geat Sly Mla Sly Gin Asp '110 gca gga ggg toe Ala GlY Sly Ser ttg ago Lou Ser gag ctq tta GinU LoU Len 9 04
S
9 .5 9 .5
S
agg cog t Arg Pro Phe .130 cag gos atg Gin. Ala met aet ttt aca ggt atq aco tat ttg.ccq Asn Who Thr.Sly met Thx Tyr Loeu Pro 140 ttc gog Ctg tao Pbe Ala Lou Tyr gut Gly 150 gta eat ggg gcg Val Aen Sly Ala gat 909. acg gat Asp -Ala Thr Asp 155 att cat Il. His .160.
gao eat gcc aaa Asp Asn*Ale Lys ctg got got tao Lieu Ala Ala Tyr eta Ile 170 aeg aaa toe ttt ta Lye Lye 8cr Phe
S
9 S S 9* 9
S
*95* 9* **qS 9 <210> 2 <211> 174 <212> PRT <213> Bacillug anmyioliqiefacijna '400> 2 Met Lye Val. Leu Val. Lou Ala Val His Pro 1 -5 10 Asp Met Gin Aen Sex- Ala Val. Aen Lys Ala Tx-p Ala Gin Gin 1Lou 2.5 Lys Lys His Asp Gin Len Thr Ile ASP Ala Gin Va. Arg Gin neu Tyr *ye bl *ayr Pro Asp Swy sin 40 4.
COMS IDNo:SBMI-00906201 Received by IP Australia: lime 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACK +61 2 99255911 0628337999 m55 Dl ND. 525 0019 Lys Gin -Arg Gin Le-a CYs Giu Gin tryr Asp Arglev eGiPe S0 55 Il l h 3ppe Pro Leu Tryr ?z-p Tyr Ser Ala Pro Pro Lou Leu-Lye Thr Trp Mea.t Asp 70 75 His Val Len Ser Tyr G;ly Trp' Ala Tyr'Cly Ser Lys G .ly Lys Ala Leu His GlyLyB3 Gin LCU.Met-Leu Ala Val Ser Val. Cly Ala.l i s 100 105 110Cu Ala Tyr Gin Ala Gly Gly Ser Asenpia Phe Thx Len Ser GlU Leu LSU 115 120 125 Arg Pro PMe GinI Ala Met Ala Aen Phe Thr.Gly MetThr Tyr Leu Pro 130 135 140 0*Se*9
S
S
is S S 5 0 0*e 5.* 0e
S
S
5* S S
S.
S
5* 0*
S.
*550
S
S
Ala Phe Ala 145 Len Tyr Gly Va. Asn Gly Ala Asp Ala Tbr Asp 150 155 Ile His 160 ASP AMn Ala Lys Arg Len Ala 165 Ala Tyr Ile Lye Lye Ser Phe 170 C210> a <211> 528 <212> DNAj 213> Bacillus subtiiis c220> <221> c!Ds <222> (28 <223> <400> 3 erg aaa ata ttg gtt Met Lys le Len Val 1 5 ttg gca gtg cat Leu Ala Val His cct pro cat atg gag acc His Met Gin Thr tca gtt Sex- Val 9tt aat aag Val Asn Lys 7 gcg Ala, tgg gct gag gaa Trp Ala Gin Gin agt aaa. cat gjac Ser Lye His Asp Bat ato aca Asn Ile Thr COMS ID No: SBMi-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACK +61 2 99255911 4 062837999N.55 02 NO.525 0020 gta. cgg gat ctt Val Arg Asp Leu aag gaa cay cay Lye Gin Gin .0-n so cy eta tat tqg Pro Len Tyr Tz-p Ott gtg ctg act Leu Val LeU Thr Ca* t gyc &ag gag His Gly Lys Glu 100 ad tat caa geg, Lys T-yr Gin Ala 115 aaa oca. ttt C&§ Lys Pro Phe Gin 1 130 coa tat gtg ttc t Pro*Tyr Val Phe F 145 caC agt yea aaa C His S Or. Ala Lys
I
ta:
:TY~
cts Let tao Tyr tat Ctg Leu ;ge Gly Ice :at .Ir 65 taag gad tae cog gat gad 7LYS 02U -Tyr Pro Asp Gin, 40 tyc gag yea tat gat cyg CYS Glu Gin Tyr Asp Arg aqc tct cog cog etc ttg Ser Ser Pro Pro Leu Len 70 7 .99C tug get ttt gyt tca Cly Trp Ala Phe Giy Ser 90 atg ctg got gta tea ace Met Len Ala Val Ser Thr gga Sce eat cat tac tog Gly Ala Ken His Tyr Ser- 120 ecg agt eat ctg etc g9c I Tht .Ser Asn'Len Ile Gly 135 99C qtgq eat tat gca got 5 Gl.y Val Asn Tyr Ala Ala 150 155 tte gcc gad tao ae cay c Lau Ala.Gin Tyr le*Gin G 170 geg eta att Ste Ile Val aaa aaa Lye Lye Gin Gly ggq ago aiy Ser atc egt Ile Ser 125 ity eag let Lye L4 0 ~ca gag- Lie Gin ag cot t ln Pro I get As! ttt Phe tgq Trp aat Asn gaa Gin 1-10 gag Gin tat Tyr gat kep :tt ?he t gtt )Vaj.
-caa Gin Icay *Ala gca Ala Ota ren Ctq Len att Ile gtt t Val 175 909 Alat ttC Phe gat As3p ets Len.
gaa Glu ttg Eaeu clot pro :ct 'er LE0 ad 144 192 240 288 336 384 432 480 528 000000 0 000000 0 00 0 0 00 0 000 0 0000 00 0 00 0 0000 0000 0 0 0 0 0000 00 0 00 0000 0 -0 0 0000 4 00 0 00 00 0000 0 0 0 0 0000 <210>- 4 2 11> 175 c212 PRT <213> Bacillus subtilis <400> 4 Met LYe Ile Len Val Len Ala Val His Pro His Met Gin Thr 5cr Val 1 .510 Val Asn Lys Ala Trp Ala Gin Gin Len Ser Lys His Asp Asn Ile Thr 25 Vai Ary Asp ten Tyr'Lye Glu Tyr Pro Asp Gin Ala Ile.Asp Val Ala 40 COMS ID No: SBMI-00906201 Received by IPAustralia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 062837999 NO.525 Q021 Lys GIU Gin Gin Leu Cys C-1,1 Glu Tyr Asp Arg Ile Val Phe Gin Phe s0o 55 Pro Lau Tyr Tx-p Tyr Sex- Sex- pro Pro Leu Len Lye Lys Tx-p Gin Asp 70 75 8 Leu Val Len Thr Tyr sly Ta-p Ala Phe .Sly Ser Gin G1l.y Aen Ala Leu as 90 H1is Sly Lye Gin Leu Met Leu Ala Val Ser Thr sly Set Giu Ala Gl* Lys Tyr Gin Ala Sly01 l a Asn His 2WSrle e-Gn e e 1.15 120 125rIe e l LULe Lys Pro 130 Phe'cfl Ala Thr Set Aen Leu Ie 135 G1l*y Val Asn Tyr Ala
ISO
Sly Met Lys 1 *Leu- Pro 140 0 See...
*5
S.
S
5e55
SSSS
C. S S. SO 55 Pro 145 Tr Val. Phe Tyr Ala 155 Ala Gin Asp le His Sex- Ala Lys Arg Len Ala Gin Tyr Ile 170 Gin Gin Pro Phe <210> <221> 525 <212> VNA <213> Bacillns subtilis @220 <21* CD <222> <223> CD <400> atg aaa aca. tte gtt atc gtt ata cat Met Lys Thr Len Val Ile Val Ile His 1
S
aat ttg gaa. acg Asn Len Gin Thr tct gtt Sex- Val gtc aec aaa Val-Asn Lyd acc Thxt99 atg aat cgt Tx-p Net Aen Arg tta.
Len 25 aag oaa gag aaa Lys Gln Gin Lye gac att aeg Asp Ile Thx- COMS ID No: SBMI-00906201 Received by P1 Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092804 15:01 GRIFFITH HAUCK +61 2 99255911 4 062837999N.25 Q2 NO.525 0022 gtt cat gac ctg 'teea Va2. His ASP Leu Tyr ggt gaa tac cet Gly Gin Tyr Pro 40 aat ttt etc att geat 9ta gaa Asn Phe Ile- Ile A.sp Val. Gia, aaa gag Lys Gia so tag eag oto etg G1ri Gin Len Leu gat eat gag cgt Asp His Gin Arg ecC gag ttiL etc Pro Ala Len Lou, Ytt ttt cag tte- Val. Phe Gin Phe 144 240 288 atg tat tgg tee Met Tyr ?rp Tyr age agt Sar Sqr aaa, eaa tgg gaa Lys Gin Trp Gin sat stg tta ace Asp Val. Len .Thr cat gga &aa gaa His Gly Lys din 100 gat tat eaa.gea ASP Tyr Gin Al1a 115 cat Hi ggc-tgg get tat Sly.Trp Ale T'yr gga. act Sly. Thi '90 9ga gga act Giy Gly Thr aa, ttg Lys Leu eta etc tta get Len Lou Len Ala ate Ile 105 tee tea gge gee Ser Ser sly Ala eag gee tet Gin Gin Ser 110 gag ett ate Gln Len Ile 99c gga gee Sly Gly Gin eat etc *aeg ate Asn Ile Thr Ile 0.
*to.
C...0 age ceg Arg Pro 130 ttt cee gte act Phe Gin Val Thr aae tat ate gga Asn Tyr Ile Sly cgt ttt ott cet Arg Phe Len Pro ttt aca eaa tat Phe Thr Gin Tyr ggg Gly 150 ace ett eat ett Thr Leu His Len tea.
Ser 155 aaa gaa get. gtt Lys Gin Asp Val aac agt geg gag Asn Ser Ala Gin aga.
Arg 165 ttg gtt gee tat Leu Val Asp Tyr aaa gec gag eat tea Lys Ala.Gin His.
<210> 6 <211> 174 <212> PRT c213> Bacilins subtilis <400> 6 Met Lys Thr teu Vai Ile Vai Ile His Aen Len Gin Thr 5cer Val is Vel Asn Lys Thr Trp Met Asn Arg Len 25 Lys Gin Gin Lys Asp tie Thr Ile Asp Val Gin 'Val His Asp Len Tyr Sly (G1n Tyr.
40 Pro Asia Phe Ile COMS IDNo: SBMI-00906201 Received by IF Australia: Time (I-tm) 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH R-#CK +61 2 99255911 4 062837999NO55P2 NO.525 0023 Lys Glu Gin Gin Leu La Leu Asp His GIAgTeVX h i h SO 5560 Pro Net Tyr T-p, Tyr Ser Ser Pro Ala ILeu Lenu Lys GnTpl s 67075 Asp Val Lou That His Sly Ti-p Ala Tyr Gly Thr Oiy GiY Thr-Lya*Leu 90.9 His (Sly Lys Glu Leu Lieu L-en Ala 1ie Ser Seat Gly Ala Gin Glu Seat 100 .105 110 A~sp Tyr Arg Pro 130 Gin Ala Sly Gly Gin Tyr Asa Ie0 ThtleBa GnLule 115120 125 Phe Gin Val Thr Ala Asnz Tyat Ile Sly Met Arg Phle LeU Pro 135 140 0* 0 0* 0O 0e@* Ala Plie Tht Gin Tyr Sly 150 Thr Len His Len Her LYe Glu 155 Asp Val Lys 160 Aen Ser Ala Gin Arg Len Val Asp 'Tyr Len 165 170 Lys Ala Gin His <210>, 7 <211> 594 <212> DNA c2i3> Bacillus subtilis; <220> <221>
ODS
<222> 1(594) <223> <400> 7 atg gat cat atq jaa Mot Asp His Net.Lye 1 5 tee tet cgt ate eat Seat Seat Arg Ile Asn aca etc gta etc Thr Lon Val Lan gtt Val.
10 qta cat ccg aat eta gaa Va1 His Pro A-sn Ile aaa aag tgg Lys Lys Ti-p aaa Lyr gaa 9cc gtt Gin Ala Val tta agt gaa Cca Leu Seat Gin Pro 4 1 t
I-
COMS ID No: SBMI-00906201 Received by IP Australia: lime 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HCK +61 2 99255911 4 062a37999 O55 04 ND.525 IP024 C 0 0 .0 0 9 *009 .9 00** 0 0@ 9e 9* *000 0 get gta act gte cat get ott tat gaa &aa tat cgo get Ctea tea att Asp Val Thr Val ils Asp Loeu Tyr Gin Lys y r s i r i 40 45 r ApGa r l gat gtg gee ttt gee caa cacg etc ttg gOC cat gac cgt ate gtt AsV l Gi Phe Giu Gi1n Gin Gin Lou Lou Ala HBApAgleV2 so SS 60 ttt ceg ttt CCS. tta tao tgg tat ago age eta ocg ctt tta aaa tag Phe Gin Phe Pro Lou Tyrr Trp Tyr.5cr Ser Pro Pro Leu Leu Lys Gin 6570 75 t 99 ttt get gee gtg ttt aeg ttt gge tgg get cat ggt. Ccc ggc gga Trp Phe Asp Giu Val Phe Tin. Phe Gly Trp Ala His 01>' Pro Ci>' Gly 90 eat aattg aeg ggaaa gag tgg gta act gce atg teec etc ggt tca Asn Lys Lou Lye Gly Lye (flu ?r-p Vel Thr Ala Met Ser Ile*Gly Sexr 100 10510 ct gas tao tat tattcaa 9Cc ggc gga tat aa ttg ttt tog ate age Pro Gin His Ser Tyr Gin Ale Gly Gly Tyr Ann Leu P~et ger Ile Se'r 115 12012 gag etg -ace aaa ccg tto ta gce tot 9cc cat tta gte gc atg ac c Giu Lou Thr Lye Pro Phe Gin Ala gar Ala His Lou Vel Gly Met Thr 130 135 140 tat Ctg. Cot toe ttt 9Cc gee tat cgc get eat eta et& egt gaQ cae Tyr Lou Pro Bar Phe Ala. Giu Tyr Mrg Ala Asn Tin Ile Ser Asp Gin 145 ISO 155 160 gaa att gee pea agt gag sat egg tat gta aeg cat att, a eat ate Gi Ile Ala Ginu Ser Ala Asn Arg Tyr Val Lys His Ile Thr Aln Ile 165 170 175 gea tta eac cog aag gtt cge etg c egg tst~ttg aas tag ttg gag Giu Leu Aan Pro Lys Val 'Arg Leu Gin. Ag TrLn y GnLo i 1~80 18519 egt. gtc get tta saa taa 5cr Val Asp Lou Thr 195 c210> a <211> 197 <212>
PR?
<213> Bacillus subtilis <C400> 8 Met Asp His Met LysThr Leu Val, Leu Val Val His Pro Aan le Gin I S 10 144 192 240 289 336 384 432 480 529 576 594 COMS ID No: SBMI-00906201 Received by IP Australia: ime 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HA{CK +61 2 99255911 4 0G2837999 NO.525 P025 Ser Ser Arg ile As:. Lys Lys Trp Lye Giu Ala Val Leu Ser Giu Pro 25.3 Asp Val Thr Val His Asp Leu Tyr Giu Lye Tyr Ar 9 Asp Gin Pro Ile 40 Asp Val Giu- Phe Gin Gin Gin Gin Len Len Ala His Asp Arg Ile Val 50556 Phe Gin Phe Pro Leu Tyr Tip Tyr 'Ser 5cr Pro Pro Len Len Lye Gin 657075 Tr-p Phi! Asp Gin Vai Plie Thr Plie Gly Trp, Ala His Gl.yPrGyGl 90 9r5Gy l Asn Lys Leu Lys Gly Lye Gin Trp Val Thr A la Met 5cr Ile Gy Ser 100 105 110 Pro Gin His Sec Tyr Gin Ala Gly Gly Tyr An Leu phe Serle c 115 120 .125 Glu Leu Thr Lys Pro Phe, Gin Ala Ser Ala His Leu Val Gly Met Thr 130 135 140* Tryr Len Pro 5cr Phe Ala Gin Tyr Arg Ala Ann Thr Ile Ser Asp Gin 145 -150 155 160 Gin Ile Ala Gin Ser Ala Asn Arg Tyr Val Lys His lie Thr Asn Ile .5.165 170 175 S Gin Len Ash Pro Lye Val Arg Len Gin Arg Tyr Len Lye Gin Len Gin Ser Val Asp Leu Thr 195 <210> 9 <211> 630 <212> DNA 4 <2121> Bacillus subtilis 3 <220> COMS ID No: SBMI-00906201 Rec-eived by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HCK +61 2 99255911 4 062837999NO55 26 NO.525 IP026
S
S S S S 0-* 55
S
S
eS S S S S .5
S
S
<221> CDS <222> (630) <223> <400> 9 atg atc aan aCe eeC Met Ile Lys Tflr.Asn atc ego aec tat get- Sle Arg Asia Tyr Asp gag etc tta gag gee GI Ile Leu Glu Gln ece tgg cgt ttt Ctt Pro Txp) Arg*Phe Len 50 gce cog ott gee ago Ala-Pro Len Ala Ser 65 gto-ata got 9ta ttt Val Ile Ale Val Phe ato tat toe aee 900 S .le Ty'r Ser Lys Ala;i 100 gao age cee etc 9CC g Asp Arg G.in Ile Ale A 115 cag gtc eec cgt gaa a Gin Val Asn Arg Gin 130 059 ctg atg ctg act Gin Leu Met Len Thr A 145 .1 990 gga tao gat eee g (fly Sly Tyr Asp Lys G: 165 gaa ogt tat gte cog gi Gin Arg Tyr Val Pro V 180 gee ggc tat got too te Gin sly Tyr Ala 5cr 1') 195 get ttt atg gee Asp Phe met Gin cog gca gte aae Pro Ala Val Lys 25 gca eca act Ala Tbr Thr 40 gto act gae Val I1e Asp 55 ttt aao oae Phe Asn Gin 70 Ica gac atg la. Asp Met IC9 gas ctt ral Gin Leu rog cog ac .la Len Thr 120 cg atc ctg 4 hr Ile Len 1 135 ca ogo gcg C la Arg Ala E s0 ae aac etc g lu Asn Ie A :t etg ote c 11 Met Len L to ogo ctg C' rr Arg Leu P: 200 So Al.
eg' Se aca Thr aec Aen 9gt Gly 105 Ica itt le !at [is 0g l a t en 85 cg ro ace~ tat tct a Pro Ser 2cr 7cog gee gga Pro Gin Sly Cagto aca Gin Val'Thr 75 eec goa~gao Aen Me Asp teo atg cog Tyr Met Pro cat ttt gaa His Phe Gin gao. gga ggt Asp Sly sly i 140 990 tao get a GlyrTyr Asp T1 155 gaa aco ttc g Gin Thr Ph. G 170 tot etc gge a Ser Ile Sly L att get ace aa Ile Asp Thr I 2 gt C Val aee Lys ace Thr tat cag; lni lag ys :tt cea ~hr ga t ay r ae c It 9 le A
OS
atg ggc Met Lye Sly agoc ae gee Ser Lye Gin 090 Ogt tct Arg Aig Set* gee atg ace Gin Met Thr aaa gCg cag Asn Ala Gin gee eag cta Gin Lys Len tca tot get Ser 5cr Ala s0 :ta gee gee Eeu Gin Gin ;eg gtc aa 31u4 Val LYS LIb :tt cog gca .eu Pro Ala Jtt too atg rel Ser Met LaC cog ate £fl Pro Ile 160 ta gut aa en Asp Lye 175 c8 900 gao la Ae Asp Ce qea tgg la Glu Trp 48 96 144 192 240 288 336 384 432 COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09'092004 15:01 GRIFFITH HACK +61 2 99255911 4 062837?999N.25 02 NO.525 P027 aaa tea Lys <210> <211> 209 <21.2> PRT c213> Bacillus. subtilis <400> Met Ile Lye Thr Aen Asp phe met Gi Ile Met L.YS Gly Arg Arg Ser 1. 5 10 9* 9 9* .9 0 9ee* 9 9 ~0 ~0 9 99*@ 9* 9@ *9 9 9099 Ile Arg Aen Tyr Asp Pro Ala Val Lye Ile Ser Lyn GlJu 20 25 GinIle Leu Gin Glu Mla Thr Thr Ala Pro*5cr 5cr Val 35 40 45 Pro Tip Arg Phe Leu Val Ile Asp ter Pro Glu Sly Lys 50 55 60 Mla Pro Lu Mla Ser Ph6 Aeni Gin Thr Gin Val Thr Thr 65 .70 75 Val Ile Ala Val Phe Ala Asp Met'Asn Asn Ala Asp*Tyr 'as 90 Ile Tyr 5cr Lye Ala Val Gin Lau sly ?ryr Met Pro Gin 100 105 Asp Arg Gin Ile Ala Ala Leu Ti- Mla His*Phe Gin Ly's 115 .120 1;25 Gin Vai Asn Arg Gin Tin Ile Leu ILe Asp Sly Sly Lou 130 135 140 Gin L&U Met Lou Thr Ala Arg Ala His Sly Tyr Asp Tin 145 150 15 Gly Gly Tyr Asp Lys Gin Aen Ile Ala Gin Thr Phe Sly 165 170 4GUi Mot Tinr Asn Ala Gin GiU Lys Len Ser Ser Ala Len Gin Gin Gin Val Lys 110 Lou Pro Ala Val 5cr Met Aen Pro Ile 160 Len Asp Lys 17s COMS ID Na:SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACK< +61 2 99255911 4 062a37999N.55 P2 NO.525 9028 Glii Arg Tyr Val Pro V/al Net Leu Leu 5cr Ile Gly Lye AlaAlAs Glu Gly Tyr Ala Ser-Tyr Arg Leu Pro Ile Asp Thr IlAlGiTr 195 200 205 aGu r Lys <210> <211> <212 <220> <221> <222> <223 609
DNA
Bacillus subtilis '.9.999 *9999@ 9 .9 9 .9 9 0 99t* 99 0 9.
9999 9.
9 9 9999 9.
9 9 9999 Cis .(609) .<400> 11 atg acg aat Met Thr Asn tat-gat ace Tyr Asp Thr gac ctt gc Asp Leu Ala 35 ttt aca gta Phe Thr Val so COc tat eat Ala TPyr Asn tta ggc gat Leu Glly Asp act ctg gat Thr Leu Asp
S
eat UCC ccg Asn Ala Pro 20 act aaa gcg Thr Lys Ala ttc c agc Phe His Ser cee aaa caa Gin Lys Gin 70 tta aag gca Len Lye Ala gt ,t atc Ile cet Pro gat Asp 55 atc Ile eat A±sf tta aaa gca Len Lys Ala 10 t t eag gag Ser Lye Gin 25 tot 9Ct Egg 5cr Ala Trp 40 gaa tca aaa Gin Ser Lys gtt gag tct V/al Gin Ser gaa aec ggt Glu Asia Gly 90 Cgt Arg gag Glu aac Asn geg Ala tet Ser 75 gaa Glu gee Ala ctg Len ctt Len gag 'flu get Al a Vee Gin tot gta.
5cr Val act gag Thr 'flu cag cat Gin His 4S ctt. ctt.
Len Len gqtt gtt V/al Val gtt tat Val TPyr ag Lys cta Leu tg Trp cot Pro gee Ala gct Ala gaa Gin tta Leu cat His Sta V/al act Ile gee.
Gin 48 96 144 192 COMS ID No: SBMI-00908201 Received by IF Australia: lime 15:23 Date 2004-09-09 09/09/2004 15:01 09/09/204 15:01 GRIFFITH HACK +61 2 99255911 062837999NO55 02 NO.525 0029 tta gca agc esa 99c tat att Leu Ala 5cr Gin Gly Tyr Ile aeg. gat Thr Asp 1.05 gaa ate aaa cae ace ttg etc Glu Ile Lys Gin Tbr Leu Leu gge caa ate aac 99t get tat caa age gag caa ttt gcCegt gat tcc Gly Gin 110 Aan Gly Ala Tyr Gin 5cr Giu Gin Phe Ala Arg As-p 115 get tte tta Ala PhaeLeu 230 act get tet Asn*Ala Ser tta Leu 135 get gct, atg cag ctt atg att gee gea Ala Ala: Met Gin Len Met Ile Ala Ala 140 aaa gee aaa ggt tat gee act tgc gee ate gge Lye Ala Ly .s Gly.Tyr Asp Thr Cys Ala Ile Gly 245 150 155 gga ttt. &ac aaa Gly Phe Asn Lays cag ttc tea aag Gi1n Phe Gin Lys ttt gat ate Phe Asp Ile agt ga~g Ser, Gin, 170 ege tat gtt Ceg gtt atg Arg Tyr Val. Pro Val Met 175 ctt att tea Leu Ile Ser ggc gee gtg Gly Lyp Mla V'al cet geg cat aa Pro Ala His Gin age aac cgt Ser Asn Arg 528 576 609 0 000000 0 0 0 0 *000 *0 0 00 00** *000 0 0900 0* 9* 0090 0 000* ety ceg ctt tca se-a yte tea act tqg etg taa Leu Pro Len. 8cr Lye Val 5cr Th-r Tip Leu.
195 200 <210> 12 <2il> 202 <212> PR? -c213, 'Bacillus Subtilis <400> 12 Met Thr Aen Th-r LOU Asp Val Len. Lys Mla 1 5 10 Arg Ala Ser Val Lys Ohu TYr Asp Tbr Ar.n Ala Pro Ile 5cr Lys Glu 25* Gin Len Thr Glu Len Leu ASP Leu Ala Phe Thr Val so Thr Lys Ala Pro 5cr Ala Trp 40 Asn Leu Gin His Tip, His Phe His 5cr Asp Giu 5cr Lye 55 Ala Gin Leu Len Pro Val Ala Tyr Asia Gin Lys Gin 70 Ile Val Gin 5cr 8cr Ala Val Val Ala. le 75 COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092604 15:01 GRIFFITH HACK +61 2 99255911 062837999 tC55 03 NO.525 0030 Leu Gly Asp Leu Lye- Ala Asn Gin Asn Gly Gin Gin Val Tyr Ala Gin l 6590 Leu Mla 5cr Gin Gly Tyr Ile. Thr Asp Gin Ii. Lye Gin Thr Len Leu 100 10os 110 Ol ile -Aen Gly Mla 115 Tyr Gin Ser Gin Gin Phe 120 Ala Arg Asp ger 125 Ala Phe Leu Aen Ala Ser Leu Ala Mla.pMet Gin Le' Met Ile Ala Ala 130 125 14 Lye Ala Lye Gly Tyr. A ap Thrt Cys Ala Ile Gay 145 ISO 155 Gly Phe Asnx Lyg Gin Phe Gin Lys Gin Ph .e Asp Ile 5cr Gin Ar .g Tyr Val 165 170 0 *000 900* 0* 0* 0 0*@0 *0*4 0 0 .94.
090 ~0 0@ 0 0@e0 *0 ~0 0 Pro Val Met 175 Ser Asn Arg 190 Le Ile Ser Ile e 180 Gly Lye Ala Val Lys 185 Pro Ala His Gin Len Pro LeU-Ser Lye Val 5cr Thr Trp Leu 195 200 <210> 13' <211> 531.
<1>DNA <213> Escilerichia, coli <220> <22.1> CmDS <222> <223> <400> 12 atg att ctt Met Ile Leu .3.
ate att tat gog cat cog lie Ile Tyr'Ala His pro
S
tat Tyr 10 ccg cat cat tcc Pro H~is Hie 5cr cat gcg His Ala aat aaa cgg atg ctt gaa cag gca agg Asn Lys Arg Met Len. Gin Gin Ala Arg 25 acg Ctg gaa ggc Thr Len Gin Gly gtc gaa att Val Gi Ile COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 662637999 NO.525 P2031 ogo tot ott tat caa ctc tat cc, Saoc atecgtatgo c14 rr e Lou Tyr Gin Leu Tyr Pro Asp Phe Asn Il.e Asp Ile Ala Ala 3540 g g c g g g g g ctg tt cgo gcc gat ctg ato tc tgg cag cat cg19 Gin Gin Giu Ala Leu e Axg.Ala s Len Ile Val Tx-p Gin HJis pro19 50550 atg cag tgg tao ago attctco ct et aaOttg aogtaa Ma Gn ipTyr Ser Ile Pro Pro Lau Leu Lyo Len iple s y 657075 gtt ttc too cac 990 tgg got tao ggt oat ggc 940 a9 cg 0 tg cat 288 Val Phe Ser His 01/ Ti-p Ala Tyr Sly His GlIy.Gay Thr Ala Leu His 90 99c aaa cat ttg otg tgg gog gtg Bog ace g g 99ga&g a 3 Gay Lys His Leu Len W-p Mla V/al Th- Th~r Gly Sly Sly Gin Sex- His 100 .105 .110.
ttt gaa att ggt gog cat cog g9C Utt ga t gtg ctg o a o 8 P-he Gin Ile sly Ala His Pro Sly Phe ALsp te Sex sIg Pro Lena38- Sg 11.5 12; cog ttt go o 432 130 15 TrpLouPr Pro Phe Mla .a c ttattq gao gao gaa ace tc gaa 999 cag gocg 480 140 Ii s Y h h l Asp Asp Gin Thr Leu' Gin Sly Gin Ala 1 5I O155 160 S cgt cac tat aag oaa ogt ctg ctgq gaa tgg cag gag qcc cat cat g .ga 528 Arg .His Tyr Lye Gin Arg Len Len Gln Ti-p Gin Sin Ala His His Gly 165 17017 tag *5531 <c210> 14 211>* 1.7.6 <212>
PRT
213> Eacherichia coli <400> 14 Met Ie Le Ile Ile Tyr Ala His Pro Tyr Pro His Hiis Ser His Aia 1 5 10 1 Aen Lys Ax-g Net Leu Gin Gin Ala Arg Tbr Len Gin Sly Vali Gin Ile 25 Ar~g Ser Len Tyr Gin Len Tyr Pro Asp Pbs Aen tie Asp Ile Ala Ala, 40 COMS ID No: SBMI-00906201 Received by IP Australia: Time (I-tm) 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH IfCK +61 2 99255911 4 062837999 NU.525 Q032 Glu Gin Glu Ala Leu Ser Arg Ala Asp Lou Ile Val Trp Gin His -Pro so. 55 Met Gin Trp Tyr Ser Ile Pro Pro Leu Leu Lye Lou Trp Tie Asp Lye 70750 75 Val Phe Ser Rio Gly Trp Ala. Tyr G1y His (fly ly Thr Ala Leu His 90 Gdy Lye Hi* Leu Leu Trp Ala.Val Tkr Thr Gly Gly Gly lu' Ser His 100 105 110 Phe Ciu Ile Gly Ala His Pro Gly Phe Asp Val Lea Ser Gin Pro Leu 115 120 125 Gin Ala Thr Ala Ile Tyr Cys Gly Leu Aen Trp Leu Pro Pro Phe Ala 130 135 0 e 00 *e 0 @0 0 000 0 0000 00 0 0 0000* @000 0e 00 00 0 0e Met His Cys Tht*Phe Ile Cys 145 150 Asp Asp Glu Thr Leu 155 Clu Gly Gin Arg His Tyr Lye Gin Arg 165 Leu Leu Olu Gin Giu Ala His His Gly 175 <210> <211> 555 c212> DNA <213> Escherichja coli c220> <221> CDS- <222, (555) <223- <400. &tg atg tct cag Met Met Ser Gin 1 cca geg aaa gtt ttg ctg Ctg tat gcc cat ccg gaa Pro Ala LyS Val Lou Len Lou Tyr Ala His Pro Glu S 10 COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK< +61 2 99255911 4 062637999 NO.525 P?033 tot cag gac tog stg gca aac 096 sta ctS ett aaa ccg goc aeg cag96 Ser Gin Asp 5cr Val Ala Asn Arg .Val Leu Lou Lys Pro Ala Thr G;In96 2530 ote ago aat qtt. aco gtg Cac gao ott tac gog ose tat ccc gat tr144 Len Ser Aen Val Thr Val Hils Asp Leu. Tyr Ala His- Tyr Pro Asp Phe 235 *40 ttt att gat etc ccc' cgt gag cag goa tta otg oge gag cac gag gtg 192 Phe Ile Asp Ilo Pro Arg'Cau Gin Ala Leu Len Arg Gin Ris Gin Val s0 se att gte ttt eag eat oct ctt tat ace tat age tgo- cog gog eta atg 240 le Val Pho Gin Ris Pro Leu Tyr Th-r Tyr Ser Cys Pro Ala Lou Lu 70 '75 s0 aaa gag tgg ctg gee egg gta tta-agt ogt ggt ttt goc ago ggg cog 288 Lys Gin Trp .Lan Asp Arg Val Leu Ser Arg Gly Phe Ala Ser Gly Pro ena gga 9119u tqq gga aaj7a 31g *r Se a l h h *0 10S 99 ga -c a q e a g a a c t a g a c 8 130 10 4 atg cat tgg tta agt 000 ate att att tac tajg qggage egg caa ago 480 Met His.Tip Leu Ser Pro Ile Ile Use Tyr.Tr, Ala Arg Arg Gin Ser 145 ISO 155 160 S OS ga eag gag ctg gog ago ceo gotc aga g9cc tac ggt gae tgg ctg gca 528 Ala Gin Gin Len Ala Ser His Ala'Arg Mla Tyr Gly Asp Trp Leu Mla 165 170 17S aat cog otg 'tot cca oga gge ogo tga 555 Aen Pro Leu 5cr Pro Gly Gly Arg 00 421i. 18 0 1 211 184 COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACK +61 2 99255911 4* 092837999rV55P4 NO.525 (7034 Ser Gin Asp Ser Val. Ala Aan Ary Val Leu Leu Lys Pro Ala Thr Gin 25 Leu Ser Aen Val Thr Val His Asp Lou Tyr Ala His Tyr Pro ASP PIto 40 Phe Ile Asp Ile Pro Arg Glu Gin Ala Lou Len Arg*Glu His Giu Val 55 Ile Val Phe Gin 'His Pro Lou Tyr Thr T[yr Ser Cys Pro Ala Lou Leu 70 75 Lys Gin Ti-p*Leu ASP Arg Val Len Ser Arg Gly Pile Ala Ser Gly Pro 1ln Leu Al a Gly sly Asn 100 Gly Lys 0.0.00 0 00 0 ~0 0* *0ee 0 0 0* 0000 0 0* 0 0e00 Gly Glu Pro Gin Set Ala Arg 115 120 Met Ser Asp Val Len Arg Pro Ph.
130 135 Met His Trp Len Her Pro Ile Ile 145 IS0 Ala Gin Giu L en Ala 5cr His Ala 165 Asn Pro Lou Ser Pro.Gly Gly Rig <210> 17 <211> 220 <2t2> PR? <213> Haemophiiue iniuenzae ITyr Ti-p Arg 105 Tyr Asp Mla Giu Lou Mla Ile T[yr Ti-p
I
Ser Val Ile Thr Tilt 210 eSU Asn Arg T[yr Pro 125 da Gly Met eye Ara 140 Mla Arg cGiy Asp Arg Gin ser 160 TpLeu. Ala 175 Arg <400> 27 met Thr Gin Leu Thr Arg Giu Gin Val. Leu Gin Len Phe His Gin Arg 1 5 *10 is COMS ID No: SBMI-00908201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 09/09/204 15:01 GRIFFITH HCK +61 2 99255911 062837999N.55P5 NO.525 P035 Ser Thr Arg Ty~r Ty-r Asp Pro Thr Lys Lys Ile Ser Asp Gin. Asp 25 Phe Gin Cys Ile Lau Giu Cys Gily Ag Leu Ser Pro sar 5cr Va]. Gly 40 Glu so Pro Trp Lys Phe Leu 55 Val Ile Gin Aen Lys Th~r LeU Krg Gin Lys met LYS Pro Phe SerTrp Gly Met Ile An 70 75 Gin Leu Asp Aan Car His Len Val Val Ileo Leu Ala Lys Lye 90 Asn Ala Arg Tyr Asp Pro Phe phe Val Asp Val Met'Ala Arg 100 105 .Lye Gly Len Ann Ala Gin Gin Gin Gin Ala 115 Ala Len Thr LYS Tyr Lye Ala Leu 120 Gin Glu Gin Asp Met, 125 9 9 9 *99 9 9@9e 9999 9. 9 9* 9 0* 9 .999 9* 9.
*99* Lys Leu 130 Leu Gin Asfl Asp Arg Thr Lou 135 Phe Asp Tip 140 Thr GilY Ala 155 Cys 5cr Lye din Ser Ala Leu Sly 160 Thr 145 Tyr le Ala Leu Ala Asn Met Len ISO0 Asp Ser Cys Pro Ile Giu Sly.Phe His Tkr 165 170 Asp Lye Me8t Ann Gin 175 .Giu TYr Ala 'Vai 190 Cys Leu Ala Gin Giu Gly Len Phe Asp Pro Gin Car AMg Asp Val Ala Ala 195 Ile Thr Phe Sly Tyr Axg 200 Lys Ly5 eq* Arg LYS Giy ten Asp Gin 210 Va. Val Lye Trp Val c210, is <211> 205 <212> PRT <213> Therimus aguaticus COMS ID No: SBMI-00908201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092884 15:01 GRIFFITH HCK +61 2 99255911 062937999 1J55 P3 NO.525 17036 <400> 18 Met Gin Ala Thr Leu Pro Val Leu Asp Ala Lys Tht* Ala Ala Len Lys .Arg Arg 5cr Ile Arg Arg Tyr Arg Lye Asp Pro Val Pro Gin Gly Len Leu Arg Glu Ile Len Gin Ala Ala Leu Arg Ala Pro Ala Trp Asn Len Gin Pro Ti-p Arg Ile -Val.Val Val Arg Asp Ala Thr Lye .Arg Lou Arg Gin Ala Ala The 04y Gin Ala His Val Gin Glu*-Aa Val Val Leu Asp Gin Val Val Lon as Tyr Ala Asp Len Gin. Asp Ala Len Ala His Lou 90 0e His Pro Gly Val Gin Gly Gin Arg Arg Gin Ala 105 110 LYS Gin Ala Ie Gin Arg*Ala Phe Ala Ala .Met Gly Gin Gin Ala Ax-g 115120. 125 Lys Mla 130 Ti-p Ala Ser Gly Gin 135 Tyr Gly I50 Ser Tyr Ile Leu Len 140 Gly Tyi Len Len Leu Len Gin Ala Len Gly Ser Val Pro 155 Met Len Ci)' Asp Pro Giu Arg Arg Ala Ile Leu Leu Pro Ser Arg Al a Al a 175 04y Tyr Pro 190 .le Pro Ala His Arg 195 <210> 19 <211> -210 Vat Ala TLen Gly Pro Ala Gin Gin Len Pro Len Gin Arg +Val Val Len Ti-p Arg 200 COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09'9-'804 15:01 GRIFFITH HACK +61 2 99255911 062837999 tU55 13 NO.525 0037 <212> PRT <213> Synechocystis PCCSeo3 C400> 19 Met Asp Thr Pile Asp Ala Ile Tyz Gin Arg Arg Ser Val LYe His Pile Asp Pro Asp His Arg Leu Tin' Ala Giu Gln Gin Arg Lye LenHs i 25 Ala Ala Ile Gin Ala Pro Thr 3cr Pile As Ile Gin Len.
40 45 Le Ile Ile Arg Asp Pro Gin Leu*Arg Gin Thr Ii. Arg .so 55 60. Ti-p Arg Phe Glu Lys Tyr Gly Asn Gin Ala Gin Met Thr Asp Ala Her Len 70' 75 Len Ile Leu' Val 0 0 000 0 Ala Asp Val Asn Ala .Trp as ASP LYe Asp Pro Ala 90 Axrg Tyr Trp Arg Asia Mla Pro Arq Gin Val AlaL Asia Tyr Leu Val Gly Ala Ile Ala 3cr Phe 100105 110 *0*0 0 0~ 0 b.
*000 pp p ep..
p 000* p.
p p.
*000 p Tyr Gi. y Gly Lye Pro Gin Leu Gin .Arg Asp, Gin Ala Gin .115 120 *125 Arg Her Ile Met Gly Tyr Gly Met Ala Met Gin Asia Len 1230 135 Met Len Ala Ala Lye Mla 140 Asp 5cr Cye Pro Met Ile Gly Pile Asp 145 150 Lou Gin .155 Lys Val Ala Gin Val Lys Len Pro Ala Asp Tyr Ala Ile Gly Pro 165 170 Met Val Ala Ile Gly 175* Lys Arg Thr Gin Asp Ala Pro Gly Lys 180 183 Axg le Pro Leu Gly Lye Leu Leu Dye 195 200 Arg9 Arg Her Asia Her Pro Gly 190 Trp Dys Len.
Len Thr Lys Val 205 COMS ID No: SBMI-00906201 Received by IP Australia: Time (1Pm) 15:23 Date 2004-09-09 09/09/2004 09/092604 15:01 GRIFFITH HACK +61 2 99255911 4 062837999N.55Q3 NO.525 903e Ala. Ile 210 <210> <211> -172 <212> PRT <c213> Archaeoglobus fuigidus <400>* Met Gin Cys Len Asp Len Leu Phe Arg Arg Val Ser Ile Arg Lys Pine Thr Gin Asp Asp Gly Afin Mla Ala :35 Val Asp Asp Glu Ile 25 Leu Met Lys Ile Leu Gin Ala Asp Phe Val Pro Ser Ala
P.O..
P P.0.0 S. a Sly Awn 40 Lau Gin Ala Arg V/al Ile 0 s0 Arg Awn Pro Gin Lys Lye Arg Lou Mla Met Ala Ala Len Gin Met Phe Ile Gin Ala Pro Val Val le Val V/al Cys Asn Tyr Pro Arg Met Arg V/al Tyr Sly Gin Arg'.Sly Arg Len Tyr *09@ 9* ee*@ P 0* *5*0 9 Ala Gin Gin Thr Ala Leu 115 Ala Thr Ala Mla 1.o 105 Siu Awn Ile Leu Len. Ala Val.
110 Asp Gin Gin Awn Leu Sly Ala Trp Val Sly Ala Phe 125 S)~n Val 130 Ser Gin Ile Len Len Pro Glu Tyr Val.
140 Arg Pro Met Ala Ile 145 Ile Pro Ile Gly Pro Ala Gin Asn Pro 155 Ser Pro Arg Awn Tyr Pro V/al 5cr Len Thr His Phe Gin Len Trp 170 COMS ID No: SSMI-00906201 Received by IP Australia: lime 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 o62e37999 ND.525 0039 23 <210> 21 <211> 174 <212> PRT <2133. Archaeoglobus fulgidus -c400> 21 Met C3 C1 ysU G~ lu Cys eu Lys Met Ile Tr Thr Arg Ag Ser -Ile Ar ga T~c er Asp Ag Gln Ile serAPl Asp 'A 1~Le Te e 40~ILe leP~ee l Gly Asn Glu Gln Pro Trp-is phe 40 45 Ile Val Val Arg Asp Arg Glu Met Leu Lys Lys Met Ser lu Ala Pile so 55 6 Thr Phe Gly Gln Met-Leu Pro Aen Ala Ser Ala Ala Ile Val Val Cys 70, 7S 0 Ala Asp Pr Lys Leu Ser Lys Tyr p ro Tyr App Met Trp Val i s 90 GlnAs CyB.Ser Ala Ala Thr Glu Asa Ile Leu Leu Alla Ala. Arg Cys Ireu Gly i 100 105 110 Ile Oly Ser Val Trp Leu Gly Val Tyr Pro Arg Glu Glu Arg Met Lys 115 12012 Ala Leu Arg Glu LIU Leu Gly Ile Pro Glu Asxx Ile Val 'Val Phe Ser 130 135 140 Val al Ser Lteu Gly Tyr Pro LysAsp GlU Lye Asp Phe TCyr Gln Ala 14S 1501516 Asp Asp Arg Ph Asn Pro Aspp Arg Ile His Arg Olu Lys Trp 165 170 <210> 22 <211> 6o60 COMS ID No: SBM14)0906201 Received by IP Australia: Time 15:23 Date 20040909 09/09/2004 09/092004 15:01 GRIFFITH HAiCK +61 2 99255911 4 062837999 N.55 Q4 NO.S25 9040 <212> DNA <213> Campylobacter jejuni <220O> <221> CDS <222> (G06) <223> *000** 0 SOeO@* 0 I. 0 0 *000 *00e 0 00
S
0000 0 000 0*00 0* 0* *0 0 0 00e0 0* 0* 00 0000 0 c400> 22 atg Baa Baa gaa.
Met Lys Lys Gin ttt aaa eat gaa Phe Lys Aen Gin ata gca age tta Ile Ala Arg Lou 35 ata gte gtg caa.
Ile Val Val Gin 50 ant caa caa aaa Asa GIA Gin Lys '5 toa age ctt. gat 3cr Arg Lou Asp gat atg agt gaa a Asp Met 5cr Glu 100ttt tta aaa tot c Phe Leu Lye 8cr L 115 cda got cat ate g Gin, Ala [His Ile A 130 eat ata gca &gc tc Asn Ile Ala Ser qi 14£ tot tat tte toe et 8cr Tyr Lou Sor Le Ctt gaa att tt Lou Gin Ile Ph 4a c tC aaa &a Lye Lou Lys Ly ago. CcC agt tc Ser Pro Ser Se: 40 gat gag agq Asp Gin Lys Arc 55 cat gta aaa gat lie Val Lye Asp 70 :tt ttg gat tat ?he Leu Asp Tyr [5 LCa gaa atg caa 'hr Gin Met Gin ta eat caa gee en Asn Gin Gin 120 Ct ota got ago la Leu Mla 5cr 135 ?c act ata ggt (s Thr Ile Gly 150 :t get att caa in Asp Ile Gin age aca age tat tot tgt is 8cr Thr Arg Tyr 5cr CyS 10 a gag gat tta eat tot ate s Gin Asp Len Asn 3cr Ile 30 o ttg gga otg gee cot tgg r Len Gly Len Gin Pro Tip i ada gaa gaa Ott tot aae LyEs Gin Gin Leu Ser Lys tgt got. goa tta att ate ICys Ala AlaLou Ile Ile ttt gee gee aaa Ctt age Phe Gin Gin Lye Len Arg 90 an 090 tta get act tat 4 L ye Mg L6u Asp Thr Tyr b4 105 it0 cac eaa ate tot tat gca a Gin Lye Ile Ser Tyr Ala 125 e ta ctt tao agt got aat g tie -Leu Tryr Ser Ala Asn A 140 ggt. ttt get &ea gee aag o Gly Phe Asp Lye Gin Lye L.
aea gee age toe aglt ttg gi Lys Gin Arg Sor 8cr Len V~ 170 1* ag4aeat Arg Asn tta gee Lou Gin aea ttt Lye Pbs att tgc Ile Cys atc att Lea age -'YB Arg ttg cot et. pro 'ge gee '19 Gin Ct tta la Lou tt get eu Asp 160 tggtg al Val 48 96 144 1.92 240 2.88 336 354 432 480 528 COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACK +61 2 99255911 4 062237999NO55 D4 NO.525 P041 get tta gge, tat tgc aac gat aaak ace Al1a Lou Gly Tyr Cys Asn Azp Lye Lye 180 185 cat cct cas aaa aat cyt t Asn Pro Gin Ly's Aen Arg Phe &9t ttt gat gaa gtt gta aaa att avt taa Ser Phe Asp Gin Val Val Lye Ps Ile 195 200 <210> 23 c211> 201*.
c212> PRT <213> Canpylobacter jejuni c400> 23 Met Lys LYS Gin Leu Gin Ile 1 5 Phe 8cr Thr 10' Arg Tyr Ser Cys Arg Aen Phe LYe Asn Gin Lye Leu Lye Lys Gin Asp 25.
Len Asn 0 beO S 0000 0* S S a.
S
*500 000@
S
S
e.g.
50 0@ 0*S*
S
0 en.
0*
S.
S.
S.
0 Ile Ala Arg Leu Ser Pro 5cr Her Len Gly Len Giu 35 40 SerIle Len Giu Pro. Trp Lye Mhe Ser Lys Ile eye Ile Val.Val Gin Asp Gin LYS .50 55S Arg Lye Gin Gin Gin Gin Lye His Val Lys Asp.Cys Ala Mla 70 Lou Ile le Ile Ser Arg Len Asp Met Ser Phe Len Lye .115 Asp Phe Len Asp Tyr Phe Gin Sin as 90 Lys Len Are LYe Arq Thr Gin met Gin Arg Len Asp Thr Tyr Met Pro 110 Ala Arg Gin 8cr Len Asn Gin Gin Gin Lye Ile Ser Tyr 120 125 Gin Ala His 130 Ile Ala Len Ala Ser Ile Len Tyr 135 5cr 140 Ala Aen AaaLen Asn Ile Ala 5cr Cye Thr Ile Gly Gly Phe Asp 145 50 155 LYS Gin Lye Len Asp 160 COMS ID No: SBMI-00908201 Received by IP Australia: Time 15:23 Date 200)4-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 062637999 NO.525 P042 26 Ser Tyr Leu Ser Leu Asp Ile Gin Lys* Glu Arg Ser Ser eu Val Val 170 17S Al a Leu Gly Tyr Cys Asn Asp Lys Lye Aen Pro Gin Lys Asn Arg Phe 180 155 190 Ser Phe Asp Glu.Val Val Lye Phe Ile 195 200 <210> 24 <211> 52*2 <212> DNA <213> Porphyromonag 9)4givalis 221> DS .<222> <223> cc.. <400> 24 cc. atg aaaaa acg etc gta ata gte utteac ccc gat ttg ace aca toc *i Met LyE Lys Thr Leu Val Ile Va. Val His Pro Asp Leu Thr Lys -sr gtt ate aac aag get t9g qcc aaa gee atc gaa ggt gca ee act ate 96 Val Ile Aan Lys Ala Trp Ala Lye Ala -Ile Glu sly Ala Ala Thr Ile 25 0 eac cat etc tao gaa cag tat Ceg cac gga can ate gat eta ga cat 144 His His Leu Tyr Glu Gin Tyr Pro Asn sly Gin Ile Asp Leu Ala Hie gaa eaa gee ctg ctg gag gct cat gac cgc atc gt ttc caa ttc ccc 192 Glu Gin Ala Leu Leu Glu Ala His Asp Arg Ile Val Phe Gin Phe Pro so.- 50 55 60 oo6 etc tat tgg tat sa gt Ccc tat tg ctg aag aag tgg atg gc gag 240 Leu Tyr Trp Tyr Ala Ala Pro Tyr Leu LeW. Lye Lye Trp Met Asp Glu 70 75 gte ttt act gag ggc tgg goc tat ggt CC gge gga gac aag atg gag 288 Val Phe Thr Glu Sly Trp Ala Tyr Sly Ala sly Gly Asp Lye Met Glu 90 ggt aaa gaa ate tgt sea gca gtc tcc tgc gga tca ccc aaa tea get 336 sly Lye GU Ile Cys Ala Ala Val Ser eye Sly Ser Pro Lys Ser Ala 100 105 110 COMS ID No: SBMi-00905201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACK +61 2 99255911 0837999NJ55P4 NO.525 0043
III
II.
-C
.1 a.
G.e.
C. Ga
C.
C
Ge.'
C
C
C
C C C Ge
C
ttt ecC ga Phe Ala Cin 115 99a gca Cag caa Gly Ala Gin- Gin cac acg ctg His Thr Len gta ttc Val Phe 130 095 agc Arg Ser 12 095 ttC Arg Phe Sac gqg ata get ASP ely Ile Ala tac ttg sat Tyr Leu Asn ace gC tao Thr Gly Tyrttc Ctg cgc get Phe Leu Arg Ala 384 432.
480 522 cat His 145 Sc tgc tac gat Ala c 1 's Tyr Asp tac &at Oct cgC Tyr Asn- Pro ALrg etc CgC ttt ate Lou Arg Phe -le 170 Ctg Len 155 ceg gas atg Pro Gin Met ctg. ceg Len Pro 160 Uc ac tgc gas Ala Asn Cys Gin gec tat Ala Tyr 163 asa gga gaa tga Lys Gly Gin <210> <211> 173 <212>
PRT
<213> Porphyromonss Singivalis <400> Met Lys Lys 2'hr Len Val 110 Val Val His 1 510 Val 1leA Asn Lys Ala Trp Ala Ljys Ala& Ile 20 25 Pro As~p Leu Thr Lys Ser is, Gin Gly Ala Ala Thr Ile HiB His Lou T'yr Gin GinX TyrPr An l Gi le 35 40 Asp Len Ala Rig Gin Gin Mla Len Len Gin Ala His Asp Arg Ile ValPhGiPePr 50 55 h l h r Len Tyr Ti-p Tyr Ala Ala Pro Tyr Len Len Lys Lys 6570 75 Val Phe Thr Gin Gly Trp Ala Tyr Gly Ala Gly Gly 90 Gly Lys Gin Ile Cys Ala Ala Val Ser Cys Gly S er 100 105 Phe Ala Gin Gly Ala Gin Gin Cys His Thi- Le-u Arg 115 120 Trp Met Asp Gin Asp Lye Met Gin Pro Lys Ser Ala 110 Ser 12.5 Tyr Leu Asn COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 09/092804 5:01 GRIFFITH HACK +61 2 99255911 062837999NO55D4 NO.525 [?044 Val Pet Asp cay Ile Ala Ala Phe Leu.Arg Ala Ary Phe Thr Gly Tyr 130 135 140 His Ala Cys Tyr Asp Ser Tyr Aen Pro Arg Leu Pro 0Th Met Lea Pro 2,45 io155 160 Ala Aan Cys Gin Ala'Tyr Lou Arg Phe Ile Lys Sly ala 165 170 <21,0> <C211> <-212> <21.3> 26 552
D)NA
Yersinia peetis
S
S
*5
S
S
S
*5*S
*SSS
9SSS
S
*5*S
S
S.
S
S
*5*S <220> <221> CDS -<222> (1..(52 <223> <400> 26 aty atgyte Met Met Lei.
1 tCa cay gac 2cr Gin Asp tta. gaa eat Leu Gin His ttt att gat Phe Ile Asp att gta ttt .l Val Phe 6S aaa gag tgg Lys Gin Trp ggC gyc cat Gly Gly His T cag ccg ceg aag gtt tts Ct .ctg tat gee a Gn Pro Pro Lys Val -Lau Lenu Leu Tyr Ala 5. *10 tcg gtc get aac egg gtt tta ctg en& ceg 5cr Val Ala As= :Mg Val Le au Len Gin Pro 20 25 gte act gtg cac gat ctt tat yea cat tat Val Thr Val His Asp Lea Tyr Al a His Tyr 40 45 att cat cat gag Cay eaa ttg eta cyt gat Ile His His Glu ,Gin Gin Lou Leu Arg Asp 55 6 eaa cat vet tta tat act tac a~rt tgcec 0Th His Pro Lon Tyr Thr Tyr Ser Cys Pro 70 75 ttg gat egg gta agq gca cgt gyt tte gcc Lou Asp Arg Val Leu Ala Arg Gly Phe Ala 90 gca ety Beg gga aag eac tgg ege teg gtg Ala Leu Thr 0247 Lys His Trp Arg Ser V/al 100 105 cat His gta V/al c'cg Pro cat His gee Al a eat A~sn att Ile 1i10 ccg Pro is Cag Gin gat Asp Caa Gin tta Lou Gly ace Thr gaa Gin C&9 Gin ttc Phe gtt V/al cts Lou so gtt V/al ace Thr .48 96 144 192 240 288 336 COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092804 15:01 GRIFFITH HACK +61 2 99255911 062637999NO55 04 NO.525 0045 ggt gag cag Gly Gin Gin 115 a tg gaa Sgac Met Gin Asp 130 gag gga act tac Gin Gly Thr Tyr attggg gga tat le Gly Gly Tyr Cgt tac cca Arg Tyr Pro att Ct4 cyt Ile Leu Arg ttc gaa ttg acg Whe Gin Len Thr 9cg Ala 140 get &Lg tge cat Ala Met cys His.
atg Met 145 cat tgg att aat His Trp ile Abn atg att att tac Met Ile le Tyr tgg gee Trp Al a 155 aga. ego caa Arg Arg Gin ccg gaa aca etc Pro 0Th Thr Lou geeC Mla 165 agt cat gca caa Ser HIS Ala Gin get Al.a 170 tat'gtg Caa tgg Tyr Vat Gin Trp Ctg cag Len Gin 175 tea ceg etc Bar Pro Lieu aga gga etc tga Arg Gly Len <210> 27 ee**
U
U
U
U
U U
U.
U
U
<211> 183, <212> PRT i213> Yersinia petic <400> 27 Met Met La Gin Ser Gin Asp Ser 20 *Leu Gilt His Val Pro Pro 5 LYS Val LOU Leu Tyr Alit His Pro Gin V/al Ala Asn Arg Leu Lou Gin Pro Val GIn Gin Pro Asp Who Thr V/al His Len Tyr A-1a His Tyr he Ile so Ile Val
GS
Asp Ile His His Giu Gin Gin Len Lou+ Asp His Gin V/al Phe Gin His Lou Tyr Tbr Tyr Lys Gi-u Trp Lieu Asp as Arg Val Len Ala Arg Gly Cys Pro Mla Ien Leu 0 Phe Ala Asn Gly Vat Sar V/al Ile Thr Trhr 110 Gly Gly His Ala Leu Thr Gly Lys His Tip Arg 100 105 COMB ID No: SBMI-00906201 Received by IP Australia: lime 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACK +61 2 99255911 4 082837999NO55 04 NO-525 0046 Gly Gin Gin 115 Gin Gly Thx Tyr Arg Ile 120 Gly Gly Tyr Msn Arg Tyr Pro 125 met Gin 130 Met His 145 Asp Ile Leu Trp le'Asn Arg Pro 135 Phe. Gin Lon Thr.AMa Ala Met Cys His 140 Pro Met Ile Ile Tyr Trp 150 155 Ala Arg Arg Gin Pro Gin Tinr Len Set His Ala Gin Tyr Val Gin Trp Len Gin 1*75 Ser Pro Len Thr 180 Arg Gly Len
I
*9~S a a *0a~@ a a.
Ia...
<2ia> 633.
<212> DNA 213>. Helicobacter pylori <220> <221> CDS <222> .(6323) <223> <400> 28 atg aaa ttt Met Lys Phe 1 cat tct tgc His Ser Cys tta gaa gaa Len Gin Gin ttg gat Len Asp aag atgq Lys Met caa gee aaa Gin Gin Lys ttc gac ago Phe Asp Ser cee ttg eta aac Gin Len*Len Asn gag cgc Gin Arg tat gag ttt tct agt gaa gaa Tyr Gin Phe'Ser Ser Gin Giu ate got gaa ate Ile Ala Giu Ile got agg cta tog cca ago tOt tac aac Ala Arg Len Sen Pro Ser 5cr Tyr Aent 40 acy cag Thr Gi n oca tgg cat ttt Pro Trp His Phe atg gtt act aat Met Val Tin Asn gat tta aaa aa Asp Len Lys Lys COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 09/092004 15:01 GRIFFITH HACR +61 2 99255911 4 062637999tC25 04 ND.525 0047 caa att Gin Ile gca geg cac agc tat Ala Ala His Ser Ty-r ttt aat gaa gaa Phe Asen Gin Gin tdt tta aaa cce Ser Lou Lys pro 90 atg att ago (act Met Ile Lye 5cr Ala 240 288 tca gcg tta atg gtg gta.tgc Ala Len Met Val Val CYS agc gag ttg 8cr Gin Leu tta cc Leu Pro act ggc cac Thr Gly His atg caa at Met Gln Asn ctt tac cg gag tcttat aag gtt age Lou Tyr Pro Gin 3cr Tyr Lye Val. Axg 105 110 gtg ate ccc tct ttt Val Ile Pro 8cr Phe got caa atg Ala Gin Met 120 ott ggc, gtg age ttc aac cac ago.
Len Gly Val Arg Phe Asn His 125p p p pe.
S
pp p p S P *.pp atg caa .Met Gin 130 sea tta gaa agoC Lye lieu Glu 8cr tat Tyr 135 att ttg gag oaa lie Leu Oiu Gin tgc eys 140.
tat ate got gtq Tyr 1i8 Ala Val caa att tgc atg Gin Ile Cys met 99C Gly 150 gtg ago tta atg Val 2cr Len Met gga Gly 155 ttg gat agt Len Asp 5cr tgc att Cys Ile 160 gag tgt Gin Arg 175 att gga ggc ttt ile ely Gly Phe get Asp -165 cot tta aaa gtg Pro Len Lys Val ggc Gly 170 gaa 9tt. tta gaa Gin Val Len Gin 432 480 528 576 624 633 atc eat aaa Ile Asn Lys gos gjaa geg Ala Gin Ala 195 tgg ttg tg& Trp Lou 210 <210>. 29 <212.> 210 &aa at gca tgc Lye Ile A-la Cys ato get ttg Ile Ala Leu ggo aag egg gtg Gly Lye Arg Val 190 sat SOC att ac~t Asp Ala Ile Tbr 205 age tea aec toe 2cr GinLayes 8cr aga Arg 200 aaa tca aaa gtt Lys 5cr Lye Val <212> PRT <213> HeliCbbacter pylorn <400> 29 Met Lays Phe Len Asp 1 5 Gin Gin Lys Arg Phe*Asp Sen His 25 Anrg 10 Gin Len Lou Asn Gin Ang is His 3cr cys Lys Met Tyr Glu Phe 5cr Sen Gin Gin COMS ID No: SBMI-00906201 Received by IP Australia: lime 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 062837999 NO.525 0048 Leu Glu Glu Ile Ala Glu Ile Ala Arg Leu Ser Pro Ser Tyr Asn Leu Lys Lye Thr Gln so Gin Ile Trp His Phe Val Thr Asn Ala Ala His Pbe Asn Gin Ser Giu 75 Ile.Lys 2cr Ala o Gin Len Leu PrQ Ala Len Met Val Met Cys Ser Leu Pro Ser 9 9 ji.
99 999 99 999 Thr Sly His Val Ile Pro 115 Met Gin Lys Gin Asn Leu Gin Se Iyr Lys Val Arg 110 Asn His Ser Ile Ala Val Phe Ala Gin Len Glu Ser 130 Cly Gin Ile Leu Ser Leu Gly Val Arg Glu Gin Cyr 140 Met Gly Leu 155 Xl. Cys Met Asp 6cr Cye no1 Gly Sly Phe Pro Len Lye Val Ile Ala Cys Lea
I-S
dyy 170 Ile Gin Val Leu Ala Len Gly Ile Asn Lys Glu Glu Arg 175 Lys Arg Val 190 Ala lie Thr Ala Slu Ala Ser Gin Lye ser Lye Ser Lys Val Trp Leu 210 COMS ID No: SBMI-00905201 Received by IP Australia: Time 15:23 Date 2004-09-09

Claims (40)

1. A nitroreductase characterised in that it preferentially reduces CB1954 to a cytotoxic 4-hydroxylamine (4HX) derivative instead of a non-cytotoxic 2- hydroxylamine derivative, wherein the nitroreductase has at least 70% identity with a sequence selected from the group consisting of SEQ ID Nos: 2, 4, 12, 17, 20, 21, 23, 25 and 29.
2. A nitroreductase according to Claim 1, wherein the nitroreductase has at least 90% identity with a sequence selected from the group consisting of SEQ ID Nos: 2, 4, 10, 12, 17, 20, 21, 23, 25 and 29.
3. A nitroreductase according to Claim 1, wherein the nitroreductase has at least 95% identity with a sequence selected from the group consisting of S 15 SEQ ID Nos: 2, 4, 10, 12, 17, 20, 21,23, 25 and 29.
4. A nitroreductase according to Claim 1, wherein the nitroreductase has a 0.*i sequence selected from the group consisting of SEQ ID Nos: 2, 4, 10, 12, 17, 20, 21, 23, 25 and 29.
5. A nitroreductase according to any one of Claims 1 to 4, further characterised in that it reduces CB1954 to the 4HX derivative with a Km of less than 700 micromolar. 25 6. A nitroreductase according to any one of Claims 1 to 5 further characterised in that it is NADPH dependant.
7. A nitroreductase according to any one of Claims 1 to 6, further characterised in that it reduces CB1954 to a cytotoxic 4-hydroxylamine (4HX) derivative substantially without producing the non-cytotoxic 2-hydroxylamine derivative. COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 062837999 NO.525 D011
8. A nitroreductase according to any one of Claims 1 to 7 which reduces the prodrug to the toxic derivative with a Kcat of at least 8.
9. A nitroreductase according to any one of Claims 1 to 8, which reduces CB1954 or an analogue thereof to a toxic derivative, shares at least sequence identity with the rat DTD sequence and does not contain a domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence.
10. A nitroreductase characterised in that it reduces a prodrug to a toxic derivative with a Km of less than 700 micromolar, wherein the prodrug is selected from CB1954 and analogues thereof, and wherein the nitroreductase has at least 70% identity with a sequence selected from the group consisting of SEQ ID Nos: 2, 12, 17, 20, 21 and 23:
11. A nitroreductase according to Claim 10, wherein the nitroreductase has at least 90% identity with a sequence selected from the group consisting of SEQ ID Nos: 2, 12, 17, 20, 21 and 23.
12. A nitroreductase according to Claim 10, wherein the nitroreductase has at least 95% identity with a sequence selected from the group consisting of SEQ ID Nos: 2, 12, 17, 20, 21 and 23. t
13. A nitroreductase according to Claim 10, wherein the nitroreductase has 25 a sequence selected from the group consisting of SEQ ID Nos: 2, 12, 17, 20, 21 and 23.
14. A nitroreductase according to any one of Claims 9 to 13 which reduces the prodrug to the toxic derivative with a Km of less 300 micromolar. A nitroreductase according to any one of Claims 9 to 14 which reduces the prodrug to the toxic derivative with a Kcat of at least 8. COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 062837999 N0.525 D012 26
16. A nitroreductase according to Claim 15 which reduces the prodrug to the toxic derivative with a Kcat of at least
17. A nitroreductase according to any one of Claims 9 to 16, further characterised in that it reduces CB1954 to a toxic derivative, it reduces SN23862 to a toxic derivative, it can use both NADH and NADPH as electron donor and in that it shares no more than 30% sequence identity with the E.coli NfnB sequence.
18. A nitroreductase according to any one of Claims 9 to 17 further characterised in that it shares at least 50% identity with the rat DTD sequence and in that it does not contain a domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence. 15 19. A nitroreductase characterised in that it reduces a prodrug to a toxic derivative with a Kcat of at least 8, and wherein the nitroreductase has at least identity with a sequence selected from the group consisting of SEQ ID S' Nos: 10, 12, 17, 20, 21 and 23.
20. A nitroreductase according to Craim 19, wherein the nitroreductase has at least 90% identity with a sequence selected from the group consisting of SEQ ID Nos: 10, 12, 17, 20, 21 and 23. o
21. A nitroreductase according to Claim 19, wherein the nitroreductase has 25 at least 95% identity with a sequence selected from the group consisting of SEQ ID Nos: 10, 12, 17, 20, 21 and 23.
22. A nitroreductase according to Claim 19, wherein the nitroreductase has a sequence selected from the group consisting of SEQ ID Nos: 10, 12, 17, 21 and 23.
23. A nitroreductase according to any one of Claims 18 to 22, further characterised in that it reduces CB1954 to a toxic derivative, it reduces COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 062837999 N0.525 0013 27 SN23862 to a toxic derivative, it can use both NADH and NADPH as electron donor and in that it shares no more than 30% sequence identity with the E.coli NfnB sequence.
24. A nitroreductase according to any one of Claims 18 to 23, further characterised in that it reduces CB1954 or an analogue thereof to a toxic derivative, in that it shares at least 50% sequence identity with the rat DTD sequence and in that it does not contain a domain that is the same as or corresponds to amino acids 51 to 82 of the rat DTD sequence. A nitroreductase characterised in that it reduces CB 1954 to a toxic derivative, it reduces SN23862 to a toxic derivative, it can use both NADH and NADPH as electron donor and in that it shares no more than 30% sequence identity with the E.coli NfnB sequence, and wherein the nitroreductase has at 15 least 70% identity with a sequence selected from the group consisting of SEQ SID Nos: 10, 12, 17 and 29.
26. A nitroreductase according to Claim 25, wherein the nitroreductase has at least 90% identity with a sequence selected from the group consisting of 20 SEQ ID Nos: 10, 12, 17 and 29.
27. A nitroreductase according to Claim 25, wherein the nitroreductase has S: at least 95% Identity with a sequence selected from the group consisting of SEQ ID Nos: 10, 12, 17 and 29.
28. A nitroreductase according to Claim 25, wherein the nitroreductase has a sequence selected from the group consisting of SEQ ID Nos: 10, 12, 17 and
29. 29. A nitroreductase according to any one of claims 24 to 28, wherein the sequence identity with the Ecoli NfnB is about 25% or less. ri: COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 062837999 N0. 525 0014 28 Use of a DNA sequence coding for a nitroreductase according to any preceding Claim in manufacture of a medicament for prodrug therapy.
31. A viral vector, comprising a DNA encoding nitroreductase according to any one of Claims 1 to 29 operatively coupled to a promoter for expression of the DNA.
32. A mini-gene comprising 10 a DNA encoding nitroreductase according to any one of Claims 1 to 29 operatively coupled to a promoter for expression of the DNA.
33. A pharmaceutical composition comprising a nitroreductase according to S. 15 any one of Claims 1 to 29 in combination with a pharmaceutically acceptable carner. o
34. A pharmaceutical composition for use in a directed-enzyme prodrug therapy, comprising a pharmaceutically acceptable carrier and a compound for 20 converting a prodrug into a drug, wherein a compound comprises a ntroreductase according to any one of Claims 1 to 29 conjugated to a targeting :o moiety.
35. A pharmaceutical composition according to Claim 34 wherein the 25 targeting moiety comprises an antibody specific for a target cell.
36. A pharmaceutical composition according to Claim 34 wherein the targeting moiety Is a moiety preferentially accumulated by or taking up by a target cell.
37. A method of preparing a nitroreductase, comprising expressing a gene in a bacterial cell, wherein the gene codes for a nitroreductase according to any one of Claims 1 to 29. COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH -HCK +61 2 99255911 4 062837999 N0.525 0015 I 29
38. Use of a nitroreductase according to any one of Claims 1 to 29 in manufacture of a medicament for anti-tumour therapy.
39. Use of a compound comprising a nitroreductase according to any one of Claims 1 to 2 9 conjugated to a targeting moiety in manufacture of a medicament for anti-tumour therapy. A method of treating cancer comprising administering a therapeuticlly effective amount of a nitroreductase according to any one of Claims I to 29, a composition according to any one of Claims 33 to 36, a viral vector according to Claim 31, or a mini-gene according Claim 32. lc41. Use of a nitroreductase having at least 70% identity with a sequence oo:" ~15 selected from SEQ ID Nos: 2, 4, 10, 12, 17,20, 21, 23, 25 and 29, for the manufacture of a medicament for prodrug therapy. S42 Use according to Claim 41' wherein the nitroreductase preferentiall reduces CB1954 to a cytotoxic 4 -hydroxylamine (4HX) derivative instead of a S. 20 non-cytotoxic 2 -hydroxylamine derivative.
43. Use according to Claim 41 or 42, wherein the nitroreductase has at ^20, 21,23, 25 and 29. S
44. Use according to Claim 41 or 42, wherein the nitroreductase has at least 95% identity with a sequence selected from SEQ ID Nos: 2, 4, 10, 12, 17, 21, 23, 25 and 29
45. Use according to Claim 41 or 42, wherein the nitroreductase has a sequence selected from SEQ ID No: 2, 4, 10, 12, 17, 26, 21, 23, 25 and 29. COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004-09-09 09/09/2004 15:01 GRIFFITH HACK +61 2 99255911 4 062837999 NO.525 P~016 described with reference to any one of the Examples.
47. A nitroreductase according to Claim 10 substantially as hereinbefome described with reference to any one of the Examples.
48. A nitroreductase according to Claim 19 substantially as hereinbefore described with reference to any one of the Examples.
49. A nitroreductase according to Claim 25 substantially as hereinbefore described with reference to any one of the Examples. Dated this 9th day of September 2004 13y their Patent Attorneys GRIFFITH HACK COMS ID No: SBMI-00906201 Received by IP Australia: Time 15:23 Date 2004409-09
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US10357577B2 (en) 2010-07-16 2019-07-23 Auckland Uniservices Limited Bacterial nitroreductase enzymes and methods relating thereto
GB201320679D0 (en) 2013-11-22 2014-01-08 Univ Nottingham Polypeptides,polynucleotides and uses thereof
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EP0540263A1 (en) * 1991-10-23 1993-05-05 Cancer Research Campaign Technology Limited Bacterial nitroreductase for the reduction of CB 1954 and analogues thereof to a cytotoxic form

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EP0540263A1 (en) * 1991-10-23 1993-05-05 Cancer Research Campaign Technology Limited Bacterial nitroreductase for the reduction of CB 1954 and analogues thereof to a cytotoxic form

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