US20020045739A1 - Acyl glucosaminyl inositol amidase family and methods of use - Google Patents

Acyl glucosaminyl inositol amidase family and methods of use Download PDF

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US20020045739A1
US20020045739A1 US09/733,569 US73356900A US2002045739A1 US 20020045739 A1 US20020045739 A1 US 20020045739A1 US 73356900 A US73356900 A US 73356900A US 2002045739 A1 US2002045739 A1 US 2002045739A1
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amidase
mycothiol
ala
conjugate
amino acid
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Gerald Newton
Yossef Av-Gay
Robert Fahey
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CALIFORNIA SAN DIEGO THE, University of, Regents of
University of British Columbia
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)

Definitions

  • the present invention generally relates to a family of enzymatic compounds produced by bacteria and methods of their use in drug discovery and degradation of toxic substances, and more specifically to acyl glucosaminyl inositol amidases and methods of their use.
  • Glutathione is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins (R. C. Fahey and A. R. Sundquist (1991) Adv. Enzymol.
  • Mycothiol autoxidizes more slowly than glutathione (G. L. Newton, et al. (1995) Eur. J. Biochem. 230:821-825) and mutants of Mycobacterium smegmatis defective in the biosynthesis of mycothiol have increased sensitivity to hydrogen peroxide and antibiotics relative to the parent strain (G. L. Newton, et al. (1999) Biochem. Biophys. Res. Commun. 255:239-244).
  • This observation suggests that mycothiol may play a key role in the protection of actinomycetes against oxygen toxicity and reactive toxins.
  • the biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.
  • a mycothiol homolog of glutathione reductase was recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al. (1999) J. Amer. Chem. Soc. 120:11538-11539, M. P. Patel, et al. (1999) Biochem. 38:11827-11833).
  • the reductase is reasonably specific for the disulfide of mycothiol but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol (M. P. Patel, et al. (1999) supra.). Therefore, there is a need in the art for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.
  • Air, soil and groundwater in areas surrounding industrial centers and farming areas are becoming increasingly polluted with simple organic compounds with have long lifetimes in the environment.
  • These compounds include, but are not limited to 1, 2 dibromoethane, 1,2 dichloroethane, perchloroethene, trichloroethene, isoprene, and vinyl chloride. They are from pesticides, industrial degreasers, solvents, and from the production polyvinyl chloride polymers (plastics).
  • Organisms have recently been isolated from contaminated environments that have the ability to detoxify, and in some cases grow using these pollutants as a sole carbon source. There is great interest in industrialized countries in using microorganisms for biodegradation of these pollutants in soil and groundwater, a field generally known as bioremediation.
  • mycothiol is the major low molecular weight thiol and will form a mycothiol conjugate. The product of this conjugation may still be toxic.
  • Rhodococcus sp. Strain AD45 has been extensively studied for detoxification of isoprene, 1,2 dibromoethane and 1,2 dichloroethene (J. E. T. van Hylckama Vlieg, et al., Current Opinion in Microbiology, 3:257-262 (2000)).
  • the enzymes responsible for the detoxification of these toxic substances were claimed to include a glutathione S-transferase and a glutathione conjugate specific dehydrogenase (J. E. T. van Hylckama Vlieg, et al, Applied and Environmental Microbiology, 64:2800-2805 (1998); J. E. T.
  • the present invention overcomes these and other problems in the art by providing a family of purified acyl glucosaminyl inositol amidase polypeptides with enzymatic amidase activity for glucosaminyl inositol (GlcN-Ins)-containing substrates.
  • the invention acyl glucosaminyl inositol amidases are characterized by comprising an N-terminal region with an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, having four highly conserved domains wherein three of the domains contain conserved histidine residues, and having amidase activity against glucosaminyl inositol-containing amides.
  • identifying an inhibitor of acyl glucosaminyl inositol amidase activity by contacting a candidate inhibitor with an acyl glucosaminyl inositol amidase or a polynucleotide encoding the amidase in the presence of an GlcN-Ins-containing amide under suitable conditions and then determining the presence or absence of breakdown products of the amide indicative of amide hydrolase activity.
  • the substantial absence of the amide hydrolase activity indicates the candidate compound is an inhibitor of acyl glucosaminyl inositol amidase activity.
  • methods for detoxifying a toxic substance comprising contacting the toxic substance with bacteria transformed with a polynucleotide that encodes an acyl glucosaminyl inositol amidase and expressing the amidase in order to detoxify the toxic substance.
  • FIG. 1A is a drawing showing the chemical structure of mycothiol (AcCys-GlcN-Ins) (MSH), chemical name 1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy- ⁇ -D-glucopyranoside.
  • MCA mycothiol S-conjugate amidase
  • GlcN-Ins 1-D-myo-inosityl-2-amino-2-deoxy- ⁇ -D-glucopyranoside
  • AcCysR mercapturic acid
  • FIG. 1C is a schematic representation showing hydrolysis of bimane derivative (MSmB) formed by alkylation of mycothiol by monobromobimane (mBBr), which is cleaved to produce GlcN-Ins and the bimane derivative of N-acetylcysteine (AcCySmB), a mercapturic acid.
  • MSmB bimane derivative
  • mBBr monobromobimane
  • AcCySmB N-acetylcysteine
  • FIG. 2 is a schematic representation of vector pYA1082E.
  • FIG. 3 is a graph showing detoxification of mBBr by exponentially growing cells of M. smegmatis .
  • FIG. 4 is a graph showing the final step in purification of M. smegmatis amidase from Sephadex G-100 chromatography of highest specific activity fractions from Phenyl-Sepharose chromatography.
  • FIG. 5 is a schematic drawing showing MSH-dependent detoxification of mBBr or other toxins by mycobacteria.
  • FIG. 7 is a chart showing chemical structures of various substrates for mycothiol S-conjugate amidase with changes in the structure of the mycothiol moiety. MSmB activity is defined as 100%.
  • FIG. 8 is a chart showing the chemical structures of various S-conjugate substrates used to test the substrate-specificity of mycothiol S-conjugate amidase as shown in Table 2 herein. The relative activities compared with MSmB taken as 100% are shown in parentheses.
  • FIG. 9 is a chart showing alignment of the amino acid sequences of the mycothiol S-conjugate amidases of M. smegmatis mc2 155 (SEQ ID NO:1) (source TIGR) and M. tuberculosis H37Rv (Rv1082) (SEQ ID:7) (Source Sanger Center). The two sequences are 78% identical overall and the N-terminal 20 amino acids are 100% identical (SEQ ID:8).
  • FIG. 10 shows the nucleic acid sequences encoding the mycothiol S-conjugate amidase of M. smegmatis (SEQ ID NO:6) and M. tuberculosis (SEQ ID:9)
  • acyl glucosaminyl inositol amidases are characterized by having an N-terminal region with an amino acid sequence with at least 80% sequence identity to amino acid sequence MSELRLMAVHAHPDDESSKG (SEQ ID NO:2), four highly conserved domains wherein three of the domains contain conserved histidine residues, and amidase activity against glucosaminyl inositol-containing amides.
  • polypeptides have enzyme activity as an amide hydrolase and the three histidine-containing conserved regions are selected from V/F-HAHPDD (SEQ ID NO:3) of domain 1, D/HPDHINV (SEQ ID NO:4) of domain 3, and ALX-A/S-H-A/V-T/S-Q (SEQ ID NO:5) of domain 4 as shown in FIG. 6 and FIG. 9, or any combination of any two or more thereof.
  • S-conjugate amidases A subset of the acyl glucosaminyl inositol amidases are referred to herein as S-conjugate amidases, whose substrate is an S-conjugate containing amide.
  • S-conjugate means that the molecule is a thioether or thioester containing two chemical moieties joined by a sulfur (i.e., —S—) moiety.
  • the S-conjugate molecule is derived from mycothiol (FIG. 1A) by the reaction shown in FIG. 1B, wherein RX is an electrophile and R is an alkyl or alkyloid moiety.
  • acyl glucosaminyl inositol amidases of the invention acyl glucosaminyl inositol amidase family do not require a sulfur-containing amide substrate and instead cleave an GlcN-Ins-containing amide substrate.
  • GlcN-Ins-containing amide and “glucosaminyl inositol-containing amide” are interchangeable when used to describe a substrate molecule for which a member of the invention family of amidases have enzymatic activity, resulting in cleavage of the molecule.
  • amide-containing S-conjugate and “S-conjugate-containing amide” are interchangeable when used to describe a substrate molecule for which a member of the invention S-conjugate amidases have enzymatic activity, resulting in cleavage of the molecule.
  • a particular member of the invention family of polypeptide amidases is an amide hydrolase
  • cleavage of the substrate molecule will form breakdown products wherein one product is a carboxylic acid, (e.g., a carboxylic acid containin at least one sulfur moiety) and the other product is a amine (e.g., GlcN-Ins).
  • the substrate is a mycothiol-derived S-conjugate amide of the type illustrated in FIG.
  • one of the breakdown products will be 1-D-myo-inosityl-2-amino-2-deoxy- ⁇ -D-glucopyranoside (GlcN-Ins) and the other breakdown product will be a sulfur-containing carboxylic acid, such as a mercapturic acid.
  • AcCys S-conjugates are termed mercapturic acids, the final excreted product in the mercapturic acid pathway of glutathione-dependent detoxification in mammals (J. L. Stevens, et al., (1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part B (D. Dolphin, et al.) pp 45-84, John Wiley & Sons, et al.).
  • invention acyl glucosaminyl inositol amidases participate in a pathway of detoxification in bacteria, especially antibiotic-producing bacteria, and that the detoxification pathway is dependent on in vivo production of a protein acyl glucosaminyl inositol amidase by such bacteria.
  • pathogenic actinomycetes that do not produce an antibiotic
  • pathogenic actinomycetes also contain a gene encoding an acyl glucosaminyl inositol amidase that becomes activated in the presence of antibiotics administered to a host, for example in treatment of a disease caused by the pathogenic actinomycetes.
  • the gene(s) encoding the invention family of amidases are a family of antibiotic-resistance genes.
  • mycothiol (1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy- ⁇ -D-glucopyranoside) (MSH) is present in a variety of actinomycetes and plays an essential role in a pathway of detoxification in such bacteria.
  • Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy- ⁇ -D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes.
  • an alkylating agent is converted to a S-conjugate of mycothiol, the latter is cleaved to release a mercapturic acid, and the mercapturic acid is excreted from the cell.
  • This process has similarities to the mercapturic acid pathway for glutathione-dependent detoxification in higher eukaryotes (J. L. Stevens, et al. (1989) supra.) but involves fewer steps.
  • S-conjugate amidase responsible for cleavage of the S-conjugate of mycothiol has been purified from M. smegmatis (SEQ ID NO:1 shown in FIG. 9) and was found to be located at amino acid residues 5717 through 4858 of a plasmid having Sanger Center Accession No. GMS-684.
  • the N-terminal region 20 residues of this newly discovered S-conjugate amidase was determined as shown in SEQ ID NO:2.
  • the nucleic acid sequence that encodes the M. smegmatis S-conjugate amidase (SEQ ID NO:6) is found at nucleic acid residues 3854 to 6717 of the plasmid having Sanger Center Accession No. GMS-684, shown in FIG. 10.
  • FIG. 9 An open reading frame encoding an identical predicted amino-terminal amino acid sequence was also identified in the M tuberculosis genome (FIG. 9).
  • the Rv1082 gene (mca) from M. tuberculosis was inserted into vector pYA1082E (FIG. 2) and expressed in E. coli , and the expressed protein was shown to have substrate specificity similar to the invention amidase from M smegmatis .
  • Homolog genes encoding S-conjugate amidases were found to be located within antibiotic synthesis operons of the antibiotic producers Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei , and the polyketide erythromycin antibiotic producer Saccharopolyspora erythraea.
  • sequence alignment also provides information that was found by screening for S-conjugate amidase homologs against bacterial genomic sequence databases.
  • M. tuberculosis an open reading frame, which encodes for homolog Rv1170, was identified.
  • mycobacteria such as M. leprae
  • a member of invention family of acyl glucosaminyl inositol amidases is encoded by ORF 05988 located in the cosmid B1740, and the M. avium homolog was represented in a contig 9 in the TIGR genome databases.
  • An additional S-conjugate amidase homolog was also identified in the M. bovis genome database that is currently underway at the Sanger Centre.
  • acyl glucosaminyl inositol amidases are formed in vivo by bacteria as part of a detoxification pathway, usually in antibiotic-producing bacteria, and most usually in bacteria characterized by intracellular production of mycothiol.
  • Mycobacterium smegmatis was treated with the alkylating agent monobromobimane (mBBr), the cellular mycothiol was converted to its bimane derivative (MSmB) (FIG. 1C).
  • the purified amidase (0.044 ⁇ g) gave ⁇ 0.33 nmol/min/mg Cys-GlcN-Ins at a protein concentration where the amidase reaction rate for 30 ⁇ M MSmB was ⁇ 3000 nmol/min/mg.
  • the ligase reaction was also assayed for a dialyzed crude extract from M. smegmatis and 0.36 nmol/min/mg protein Cys-GlcN-Ins was formed in accord with previous reports (Newton, et al. (1999), supra., S. J. Anderberg, et al. (1998) supra.).
  • invention S-conjugate amidase does not appear to be involved in mycothiol biosynthesis since it has no significant ability to catalyze ATP-dependent ligation of cysteine with GlcN-Ins. It therefore does not appear to be a bifunctional enzyme analogous to the glutathionylspermidine synthetase/amidase which catalyzes both the biosynthesis and degradation of glutathionylspermidine in E. coli (D. S. Kwon, et al. (1997) J. Biol. Chem. 272:2429-2436) and in Crithidia fasciculata (E. Tetaud, et al. (1998) J. Biol. Chem.
  • E. coli has no mycothiol metabolism and is not expected to contain mycothiol conjugate amidase endogenous proteins that would give background to these assays.
  • the amidase activity of the M. tuberculosis -derived S-conjugate amidase expressed in E. coli was found to be associated with the insoluble cell pellet material.
  • MSmB 0.1 mM MSmB as substrate, the resolublilzed crude protein extract was found to produce 4.1 ⁇ 0.05 nmoles/min/mg protein GlcN-Ins and 5.4 ⁇ 0.3 nmoles/min/mg protein AcCysmB.
  • acyl glucosaminyl inositol amidases participate in detoxification of antibiotics or the antibiotic biosynthesis by-products in actinomycetes and other bacteria.
  • pathogenic bacteria e.g., bacterial infections
  • therapeutic antibiotics administered to the subject being treated may have limited effectiveness in treating the disease because of innate resistance of the pathogenic bacterium to antibiotics subject to such a detoxification pathway.
  • Such a bacterium may prove resistant to the therapeutic antibiotic administered to the subject hosting the bacterium.
  • pathogenic bacteria particularly susceptible to such resistance are pathogenic actinomycetes, such as those derived from M. smegmatis, M. tuberculosis, M. leprae, M. bovis, Corynebacterium diphtheria, Actinomycetes israelii, M. avium , and the like, that can produce a native GlcN-Ins-containing amide.
  • identifying an inhibitor of acyl glucosaminyl inositol amidase activity by contacting a candidate compound with an acyl glucosaminyl inositol amidase or a polynucleotide encoding the amidase in the presence of an GlcN-Ins-containing amide under suitable conditions and then determining the presence or absence of breakdown products of the amide indicative of amide hydrolase activity.
  • the substantial absence of the amide hydrolase activity is indicative of a compound that inhibits activity of the amidase.
  • the candidate compound is an inhibitor of the S-conjugate amidase.
  • the inhibitor may be a polypeptide, oligonucleotide, or small molecule.
  • the inhibitor is a compound, such as an antisense oligonucleotide, that inhibits intracellular production of the amidase.
  • the antisense oligonucleotide can be complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with at least 80% sequence identity to the amino acid sequence of SEQ ID NOS:2, 3, 4 or 5 and conservative variations thereof.
  • the antisense oligonucleotide hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.
  • the candidate compound inhibits intracellular production or activity of the acyl glucosaminyl inositol amidase.
  • a presently preferred drug candidate for screening in live bacteria for activity that inhibits intracellular production or activity of acyl glucosaminyl inositol amidase is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence with at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, or a conservative variation thereof, for example, 85%, 90%, 95% or 100% sequence identity.
  • Suitable conditions for conducting invention drug screening methods are well known in the art and are described, for example, in the Examples hereinbelow.
  • the inhibitor can be an antisense oligonucleotide complementary to a target region in a messenger RNA that encodes an acyl glucosaminyl inositol amidase polypeptide that is introduced into the pathogenic GlcN-Ins-amidase producing bacteria, using methods known in the art and as described herein.
  • the pathogenic bacteria can be contacted with an antisense oligonucleotide that hybridizes under intra cellular conditions with a messenger RNA that encodes an amino acid sequence segment with at least 80% sequence identity to the amino acid sequence of SEQ ID NOS:2, 3, 4, or 5 and conservative variations thereof or a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.
  • the pathogenic bacteria treated to reduce drug resistance according to the invention methods are actinomycetes, such as M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium , and the like.
  • actinomycetes such as M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium , and the like.
  • the amidase produced by the pathogenic bacteria is mycothiol S-conjugate amidase, e.g. one capable of hydrlyzing a mycothiol S-conjugate where the S-R group may be an alkyl or alkyloid group.
  • kits for increasing production of antibiotic by antibiotic-producing bacteria by contacting the antibiotic-producing bacteria with a compound that increases intracellular production by the bacteria in culture of an acyl glucosaminyl inositol amidase.
  • the increase in intracellular production of the amidase increases the production of antibiotic by the bacteria by increasing resistance of the bacteria to the antibiotic.
  • the antibiotic-producing bacteria are cultured under conditions suitable for production of the antibiotic, and the antibiotic is recovered from the culture media.
  • the compound that increases intracellular production by the bacteria of the amidase can be a polypeptide, polynucleotide, or small molecule.
  • the compound that increases intracellular production by the bacteria of the amidase is expressed intracellularly by the bacteria, preferably by actinomycetes.
  • the actinomycetes can be transformed with a polynucleotide that encodes an acyl glucosaminyl inositol and amidase and which expresses the amidase in culture.
  • Recombinant expression of the acyl glucosaminyl inositol amidase polypeptides in cultured antibiotic-producing cells can be useful for increasing the resistance of the production cells to the toxic effect upon themselves of the antibiotics they produce.
  • Suitable polynucleotides that can be used to transform antibiotic-producing bacteria can contain nucleic acid residues 34318-35184 of the polynucleotide having GenBank Accession No. gi2896719 or encode a polypeptide containing amino acid residues 5717-4858 of Sanger Center Accession No. 684.
  • Suitable bacteria for use in the invention method for increasing production of antibiotics by antibiotic-producing bacteria include Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei and Saccharopolyspora erythraea.
  • the bacteria is a strain currently in use for detoxification of environmental pollutants and the bacteria are transformed with a polynucleotide that encodes the amidase such that the amidase is expressed intracellularly under environmental conditions.
  • the environmental condition may include or be a pollutant.
  • Such environmental pollutants that may be detoxified according to invention methods include, but are not limited to 1, 2 dibromoethane, 1,2 dichloroethane, perchloroethene, trichloroethene, isoprene, and vinyl chloride. They are from pesticides, industrial degreasers, solvents, and from the production polyvinyl chloride polymers (plastics), such as a halogenated hydrocarbon, or the epoxides, such as isoprene monoxide, and the like.
  • plastics such as a halogenated hydrocarbon
  • epoxides such as isoprene monoxide, and the like.
  • the amidase cleaves the N-acyl glucosaminyl inositol, freeing GlcN-Ins as one of the cleavage breakdown products.
  • GlcN-Ins has utility in conducting research regarding amidase activity and mycothiol biochemistry in bacteria, development of products and procedures for overcoming the antibiotic resistance of pathogenic bacteria, such as actinomycetes, and as a precursor for formation of acyl glucosaminyl inositol derivatives and inhibitors of amidases thereof.
  • a “conservative variation” in an amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.
  • a conservative amino acid substitution for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).
  • One or more amino acids can be deleted, for example, from an amidase polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.
  • carboxyl-terminal amino acids that are not required for amidase activity can be removed.
  • an antisense oligonucleotide can be designed to hybridize under in vivo conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2, or contains an amino acid segment as set forth in SEQ ID NOs:3, 4, or 5, or a conservative variation thereof.
  • the antisense oligonucleotide can comprises from about 10 to about 60 nucleic acid residues, for example from 10 to about 50, or from 10 to about 40, 30 or 20 nucleic acid residues.
  • “Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest will join with a complementary strand even in samples in which it is present at low concentrations.
  • Suitable intracellular conditions for hybridization of an antisense oligonucleotide to messenger RNA will be determined by the particular bacterium used in the invention method. In general, the pH, temperature and salt concentration must be comparable to intracellular conditions in the test bacterium.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
  • expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted eukaryotic genetic sequence are used in connection with the host.
  • the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
  • baculovirus vectors may also be used.
  • One advantage to expression of foreign genes in this invertebrate virus expression vector is that it is capable of expression of high levels of recombinant proteins, which are antigenically and functionally similar to their natural counterparts.
  • Baculovirus vectors and the appropriate insect host cells used in conjunction with the vectors will be known to those skilled in the art.
  • the term “recombinant expression vector” refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the invention acyl glucosaminyl inositol amidase genetic sequences. Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
  • the vector may include a phenotypically selectable marker to identify host cells which contain the expression vector.
  • markers typically used in prokaryotic expression vectors include antibiotic resistance genes for ampicillin ( ⁇ -lactamases), tetracycline and chloramphenicol (chloramphenicol acetyltransferase).
  • markers typically used in mammalian expression vectors include the gene for adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), and xanthine guanine phosphoribosyltransferse (XGPRT, gpt).
  • ADA adenosine deaminase
  • DHFR dihydrofolate reductase
  • HPH hygromycin-B-phosphotransferase
  • TK thymidine kinase
  • XGPRT xanthine guanine phosphoribosyltransferse
  • the isolation and purification of host cell expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibody.
  • Transformation of the host cell with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by electroporation or the CaCl 2 method using procedures well known in the art.
  • MgCl 2 or RbCl could be used.
  • the host used is a eukaryote
  • various methods of DNA transfer can be used. These include transfection of DNA by calcium phosphate-precipitates, conventional mechanical procedures such as microinjection, insertion of a plasmid encased in liposomes, or the use of virus vectors.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the polypeptides of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Eukaryotic host cells may also include yeast.
  • DNA can be expressed in yeast by inserting the DNA into appropriate expression vectors and introducing the product into the host cells.
  • Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene, 60:237, 1987).
  • the invention provides antibodies which are specifically reactive with invention amidase polypeptides or fragments thereof.
  • Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).
  • Monoclonal antibodies specific for acyl glucosaminyl inositol amidase polypeptide can be selected, for example, by screening for hybridoma culture supernatants which react with acyl glucosaminyl inositol amidase polypeptides, but do not react with other bacterial amidases.
  • Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).
  • antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab′) 2 and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • (2) Fab′ the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule;
  • (Fab′)2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab′) 2 is a dimer of two Fab′ fragments held together by two disulfide bonds;
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Antibodies which bind to acyl glucosaminyl inositol amidase polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired.
  • Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated herein by reference).
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the “image” of the epitope bound by the first monoclonal antibody.
  • the recombinant acyl glucosaminyl inositol amidase polypeptide is a fusion protein further comprising a second polypeptide portion having an amino acid sequence from a protein unrelated the acyl glucosaminyl inositol amidase.
  • fusion proteins can be functional in a two-hybrid assay.
  • nucleic acid having a nucleotide sequence which encodes an acyl glucosaminyl inositol amidase polypeptide, or a fragment thereof, having an amino acid sequence at least 60% homologous to one of SEQ ID NOs:2, 3, 4 or 5.
  • the nucleic acid encodes a protein having an amino acid sequence at least 80% homologous to SEQ ID NO:2, more preferably at least 90% homologous to SEQ ID NO:2, and most preferably at least 95% homologous to SEQ ID NO:2.
  • the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 40 consecutive nucleotides encoding SEQ ID NO:3.
  • the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 40 consecutive nucleotides encoding SEQ ID NO:4.
  • the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 40 consecutive nucleotides encoding SEQ ID NO:5.
  • the acyl glucosaminyl inositol amidase nucleic acid will comprise a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the acyl glucosaminyl inositol amidase-gene sequence so as to render the recombinant acyl glucosaminyl inositol amidase gene sequence suitable for use as an expression vector.
  • a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence
  • the present invention also features transgenic non-human organisms, e.g. bacteria which either express a heterologous S-conjugate amidase gene, or in which expression of their own acyl glucosaminyl inositol amidase gene expression is disrupted.
  • a transgenic organism can serve as an model for studying acyl glucosaminyl inositol amidase activity and for screening for compounds that inhibit acyl glucosaminyl inositol amidase activity in bacteria.
  • the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence encoding one of the amino acid sequences encompassed by SEQ ID NOs:2, 3, 4 or 5, or naturally occurring mutants thereof.
  • a preferred peptidomimetic which binds to an acyl glucosaminyl inositol amidase polypeptide and inhibits its binding to S-conjugate-containing amide substrate.
  • a preferred peptidomimetic is an analog of a peptide having the sequence of one of the SEQ ID NOs. 1, 2, 3, 4, or 5.
  • Non-hydrolyzable peptide analogs of such residues can be generated using, for example, benzodiazepine, azepine, substituted gama-lactam rings, keto-methylene pseudopeptides, beta-turn dipeptide cores, or beta-aminoalcohols.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • the terms “gene”, “recombinant gene” and “gene construct” refer to a nucleic acid comprising an open reading frame encoding an invention acyl glucosaminyl inositol amidase, including both exon and (optionally) intron sequences.
  • the term “intron” refers to a DNA sequence present in a given acyl glucosaminyl inositol amidase gene which is not translated into protein and is generally found between exons.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • transfection refers to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of one of the invention family of acyl glucosaminyl inositol amidases.
  • Cells or “cell cultures” or “recombinant host cells” or “host cells” are often used interchangeably as will be clear from the context. These terms include the immediate subject cell which expresses the cell-cycle regulatory protein of the present invention, and, of course, the progeny thereof. It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or difference in environment. However, such altered progeny are included in these terms, so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell. In the present case, such a characteristic might be the ability to produce a recombinant acyl glucosaminyl inositol amidase polypeptide.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • expression vector includes plasmids, cosmids or phages capable of synthesizing the subject acyl glucosaminyl inositol amidase polypeptide encoded by the respective recombinant gene carried by the vector.
  • Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
  • plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, as well as polyadenylation sites, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of a recombinant acyl glucosaminyl inositol amidase gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended.
  • the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of the regulatory protein.
  • a “transgenic organism ” is any organism, preferably a bacteria in which one or more of the cells of the organism contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus or a vector.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the transgene causes cells to express a recombinant form of the subject acyl glucosaminyl inositol amidase polypeptides.
  • transgene means a nucleic acid sequence (encoding, e.g., an acyl glucosaminyl inositol amidase polypeptide), which is partly or entirely heterologous, i.e., foreign, to the transgenic organism or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic organism or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the organism's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • the term “evolutionarily related to”, with respect to nucleic acid sequences encoding acyl glucosaminyl inositol amidase polypeptides refers to nucleic acid sequences which have arisen naturally in an organism, including naturally occurring mutants. The term also refers to nucleic acid sequences which, while derived from a naturally occurring acyl glucosaminyl inositol amidase polypeptide, have been altered by mutagenesis, as for example, combinatorial mutagenesis, yet still encode polypeptides which have the amidase activity of an acyl glucosaminyl inositol amidase polypeptide.
  • One aspect of the present invention pertains to an isolated nucleic acid comprising the nucleotide sequence encoding an acyl glucosaminyl inositol amidase polypeptide, fragments thereof encoding polypeptides having acyl glucosaminyl inositol amidase activity, and/or equivalents of such nucleic acids.
  • nucleic acid as used herein is intended to include such fragments and equivalents.
  • equivalent is understood to include nucleotide sequences encoding functionally equivalent acyl glucosaminyl inositol amidase polypeptides or functionally equivalent peptides having an activity of an acyl glucosaminyl inositol amidase polypeptide such as described herein.
  • Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will also include sequences that differ from the nucleotide sequence encoding native acyl glucosaminyl inositol amidases due to the degeneracy of the genetic code.
  • Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27° C. below the melting temperature of the DNA duplex formed in about 1 M salt) to the nucleotide sequence of an acyl glucosaminyl inositol amidase gene, such as that as set forth in nucleic acid residues 34318-35184 of GenBank Accession No. gi3256022 or the polynucleotide encoding amino acids residues 5717-4858 of Sanger Center plasmid GMS-684 (SEQ ID NO:1), particularly those segments encoding the polypeptides shown in one of SEQ ID NOs. 2, 3, 4, or 5.
  • equivalents will further include nucleic acid sequences derived from and evolutionarily related to such nucleotide sequences
  • isolated or “purified” as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.
  • an isolated nucleic acid encoding one of the subject acyl glucosaminyl inositol amidase polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the acyl glucosaminyl inositol amidase gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence.
  • isolated or purified as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • the nucleic acid of the invention encodes a peptide having an amino acid sequence as shown in GenBank Accession CAA17198 or amino acid residues 5717-4858 of Sanger Center Accession No. GMS-684.
  • Preferred nucleic acids encode a peptide having a S-conjugate amidase polypeptide activity and being at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in GenBank Accession CAA17198 (encoded by nucleic acid residues 34318-35184 of GenBank Accession No. gi3256022) or with amino acid residues 5717-4858 of Sanger Center Accession No. GMS-684.
  • Nucleic acids which encode peptides having an activity of a S-conjugate amidase polypeptide and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with such amino acid sequences are also within the scope of the invention.
  • Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes an acyl glucosaminyl inositol amidase polypeptide having all or a portion of an amino acid sequence shown in one of SEQ ID NOs. 2, 3, 4, or 5.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0 ⁇ SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 ⁇ SSC at 50° C. to a high stringency of about 0.2 ⁇ SSC at 50° C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C.
  • Isolated nucleic acids which differ from the nucleotide sequences disclosed herein due to degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject acyl glucosaminyl inositol amidase polypeptides will exist among prokaryotic cells.
  • nucleotides up to about 3-4% of the nucleotides
  • nucleic acids encoding a particular member of the acyl glucosaminyl inositol amidase polypeptide family
  • allelic variation Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
  • fragments of the nucleic acid encoding a biologically active portion of the subject acyl glucosaminyl inositol amidase polypeptides are also within the scope of the invention.
  • a fragment of the nucleic acid encoding an active portion of an acyl glucosaminyl inositol amidase polypeptide refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of, for example, the S-conjugate amidase polypeptides represented in nucleic acid residues 34318-35184 of GenBank Accession No.
  • nucleic acid fragments within the scope of the invention include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species, e.g. for use in screening protocols to detect homologs of the subject acyl glucosaminyl inositol amidase polypeptides.
  • Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant peptides.
  • a nucleic acid encoding a peptide having an activity of a S-conjugate amidase polypeptide may be obtained from mRNA or genomic DNA present in any of a number of antibiotic-producing or pathogenic bacteria, particularly actinomycetes, in accordance with protocols described herein, as well as those generally known to those skilled in the art.
  • a cDNA encoding an acyl glucosaminyl inositol amidase polypeptide for example, can be obtained by isolating total mRNA from a bacterial cell.
  • Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.
  • a gene encoding an acyl glucosaminyl inositol amidase polypeptide can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.
  • an “antisense” inhibition of endogenous production of an acyl glucosaminyl inositol amidase molecule is carried out by administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. bind) under intracellular conditions, with the cellular mRNA and/or genomic DNA encoding an acyl glucosaminyl inositol amidase polypeptide so as to inhibit expression of that protein or a constituent thereof, e.g. by inhibiting transcription and/or translation.
  • binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
  • An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the transformed cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an acyl glucosaminyl inositol amidase polypeptide.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding one of the subject acyl glucosaminyl inositol amidase proteins.
  • Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo.
  • Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense techniques have been reviewed, for example, by van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.
  • the oligomers of the invention may be used as reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.
  • This invention also provides expression vectors comprising a nucleotide sequence encoding a member of the invention family of acyl glucosaminyl inositol amidase polypeptides and operably linked to at least one regulatory sequence.
  • Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
  • Regulatory sequences are art-recognized and are selected to direct expression of the peptide having an activity of an acyl glucosaminyl inositol amidase polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements.
  • exemplary regulatory sequences are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the acyl glucosaminyl inositol amidase polypeptides of this invention.
  • Such useful expression control sequences include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast .alpha.-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • T7 promoter whose expression is directed by T7 RNA polymerase
  • the major operator and promoter regions of phage lambda the control regions for fd coat protein
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • the subject gene constructs can be used to cause expression of the subject acyl glucosaminyl inositol amidase polypeptides in cells propagated in culture, e.g. to produce proteins or peptides, including fusion proteins or peptides, for purification.
  • recombinant expression of the subject acyl glucosaminyl inositol amidase polypeptides in cultured antibiotic-producing cells for example during large-scale production of antibiotics by antibiotic-producing bacteria, can be useful for increasing the resistance of the production cells to the toxic effect upon themselves of the antibiotics they produce.
  • the level of antibiotics in the culture media can be increased without causing death of the production cells, thereby increasing the efficiency of industrial antibiotic production methods.
  • This invention also pertains to a host cell transfected with a recombinant acyl glucosaminyl inositol amidase gene in order to express a polypeptide having an activity of an acyl glucosaminyl inositol amidase polypeptide.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • an acyl glucosaminyl inositol amidase polypeptide of the present invention may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
  • Another aspect of the present invention concerns recombinant acyl glucosaminyl inositol amidase polypeptides which are encoded by genes which have the amidase activity of an acyl glucosaminyl inositol amidase polypeptide, or which are naturally occurring mutants thereof.
  • the term “recombinant protein” refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the acyl glucosaminyl inositol amidase polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
  • phrase “derived from”, with respect to a recombinant gene encoding the recombinant acyl glucosaminyl inositol amidase polypeptide is meant to include within the meaning of “recombinant protein” those proteins having an amino acid sequence of a native acyl glucosaminyl inositol amidase polypeptide, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions of a naturally occurring acyl glucosaminyl inositol amidase polypeptide of a organism.
  • the present invention further pertains to methods of producing the subject acyl glucosaminyl inositol amidase polypeptides.
  • a host cell transfected with expression vector encoding one of the subject acyl glucosaminyl inositol amidase polypeptide can be cultured under appropriate conditions to allow expression of the peptide to occur.
  • the peptide may be secreted and isolated from a mixture of cells and medium containing the peptide. Alternatively, the peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • the peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the subject acyl glucosaminyl inositol amidase polypeptides.
  • nucleotide sequence derived from the cloning of an acyl glucosaminyl inositol amidase polypeptide of the present invention encoding all or a selected portion of the protein, can be used to produce a recombinant form of the protein via microbial cellular processes.
  • the recombinant acyl glucosaminyl inositol amidase polypeptide can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in bacterial cells.
  • Expression vehicles for production of a recombinant acyl glucosaminyl inositol amidase polypeptide include plasmids and other vectors.
  • suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein).
  • These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
  • drug resistance markers such as ampicillin can be used.
  • baculovirus expression systems include pVL-derived vectors (such as pVL 1392, pVL 1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the .beta.-gal containing pBlueBac III).
  • pVL-derived vectors such as pVL 1392, pVL 1393 and pVL941
  • pAcUW-derived vectors such as pAcUW1
  • pBlueBac-derived vectors such as the .beta.-gal containing pBlueBac III.
  • This invention further contemplates a method of generating sets of combinatorial mutants of the present acyl glucosaminyl inositol amidase polypeptides, as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs) that are functional in cleaving S-conjugate amide molecules.
  • potential variant sequences e.g. homologs
  • the amino acid sequences for a population of acyl glucosaminyl inositol amidase polypeptide homologs are aligned, preferably to promote the highest homology possible.
  • Such a population of variants can include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation.
  • Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.
  • the presence or absence of amino acids from an aligned sequence of a particular variant is relative to a chosen consensus length of a reference sequence, which can be real or artificial.
  • deletions in the sequence of a variant relative to the reference sequence can be represented by an amino acid space (*), while insertional mutations in the variant relative to the reference sequence can be disregarded and left out of the sequence of the variant when aligned.
  • Xaa(1) represents Ser, Thr, Asn or Gln
  • Xaa(2) represents Gly, Ala, Val, Leu, or Ile
  • Xaa(3) represents Arg, Lys or His
  • Xaa(4) represents Gly, Ala, Val, Leu, Ile, Asp or Glu
  • Xaa(5) represents Gly, Ala, Val, Leu, Ile, Asn or Gln
  • Xaa(6) represents Arg, Lys, His, Tyr or Phe
  • Xaa(7) represents Asp or Glu
  • Xaa(8) represents Pro, Gly, Ser or Thr
  • Xaa(9) represents Gly, Ala, Val, Leu, Ile, Asp or Glu
  • Xaa(10) represents Gly
  • amino acid replacement at degenerate positions can be based on steric criteria, e.g. isosteric replacement, without regard for polarity or charge of amino acid sidechains.
  • completely random mutagenesis of one or more of the variant positions (Xaa) can be carried out.
  • the combinatorial acyl glucosaminyl inositol amidase library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential acyl glucosaminyl inositol amidase polypeptide sequences.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential acyl glucosaminyl inositol amidase nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display) containing the set of acyl glucosaminyl inositol amidase polypeptide sequences therein.
  • the library of potential acyl glucosaminyl inositol amidase homologs can be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression.
  • the purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential acyl glucosaminyl inositol amidase sequences.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of acyl glucosaminyl inositol amidase homologs.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene product was detected.
  • Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.
  • the invention also provides for reduction of the subject acyl glucosaminyl inositol amidase polypeptides to generate mimetics, e.g. peptide or non-peptide agents, which are able to mimic binding of the authentic acyl glucosaminyl inositol amidase polypeptide to a substrate S-conjugate amide molecule.
  • mimetics e.g. peptide or non-peptide agents
  • Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of an acyl glucosaminyl inositol amidase polypeptide which participate in protein-protein interactions involved in, for example, binding of the subject acyl glucosaminyl inositol amidase polypeptide to a substrate.
  • the critical residues of a subject acyl glucosaminyl inositol amidase polypeptide which are involved in molecular recognition of substrate can be determined and used to generate acyl glucosaminyl inositol amidase-derived peptidomimetics which bind to S-conjugate amide substrates and, like the authentic acyl glucosaminyl inositol amidase polypeptide, cleave the substrate molecule, for example by amide hydrolase activity.
  • peptidomimetic compounds e.g. diazepine or isoquinoline derivatives
  • non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.
  • Glutathione S-conjugates are exported to the plasma and transported to other tissues, notably kidney and liver, where they are extracellularly degraded by y-glutamyltranspeptidase to CySR-Gly and the latter cleaved by a dipeptidase to produce a cysteine S-conjugate, CySR (J. L. Stevens, et al. (1989) supra.). CySR is imported and acetylated by acetyl CoA to produce a mercapturic acid (AcCySR) which is ultimately excreted in urine and bile.
  • AcCySR mercapturic acid
  • Glutathione-producing cells excrete the bimane derivative of glutathione (GSmB) intact.
  • E. coli A. Kaluzna, et al. (1977) Biochem. Mol. Biol. Int. 43:161-171
  • yeast Z. Li, et al. (1996) J. Biol. Chem. 271:6509-6517)
  • plants E. Martinola, et al. (1993) Nature 364:247-249
  • cultured mammalian cells R. P. J. Oude Elferrink, et al. (1993) Hepatology 17:434-444, T. Ishikawa, et al. (1994) J. Biol. Chem.
  • MSmB was the best of the substrates tested and modification of the bimane by attachment of a positively charged trimethylammonio group (MSqB) or a negatively charged p-sulfobenzoyloxy residue (MSsB) led to a 6-7-fold loss of activity (FIG. 8).
  • MSC cerulenin
  • Invention S-conjugate amidase has little activity with mycothiol or mycothiol disulfide (FIG. 8), which is an essential specificity in order to minimize a futile cycle involving amidase degradation of mycothiol or mycothiol disulfide in combination with mycothiol biosynthesis.
  • mycothiol is not a substrate for the amidase, at mM levels it does inhibit amidase activity with MSmB as substrate.
  • the thiol biotinylating reagent 3-(N-maleimidopropionyl)biocytin (MPB) is utilized to capture mycothiol as the MSMPB conjugate in our current immunoassay protocols for determination of mycothiol (M. D. Unson, et al. (1999) J. Clin. Microbiol. 37:2153-2157). Since MSMPB is a substrate for invention S-conjugate amidase (Table 2, FIG. 8), it is important that the amidase be inactivated when assaying cells by use of protein denaturing conditions for cell extraction as employed here and in the earlier study (M. D. Unson, et al. (1999) supra.).
  • the invention family of acyl glucosaminyl inositol amidases is an important practical tool for studies of mycothiol biochemistry because it provides an efficient means for producing GlcN-Ins.
  • GlcN-Ins is required as a substrate for the assay of ATP-dependent cysteine:GlcN-Ins ligase (S. J. Anderberg, et al. (1998) supra., C. Bornemann, et al. (1997) supra.), as a standard for HPLC calibration (S. J. Anderberg, et al. (1998) supra.), and as a precursor of synthetic analogs.
  • Mycothiol is easily isolated from M.
  • the high mycothiol content relative to the glutathione content of this organism along with the inability to saturate the glutathione S-transferase with the substrate glutathione suggests that the enzyme used by mycobacteria to detoxify toxic environmental substances, such as vinyl chloride, 1,2 dibromoethane, numerous other haloalkanes, and the like, is actually a mycothiol S-transferase and not a glutathione S-transferase.
  • Mycothiol S-conjugate amidase is present in the test organism (at levels higher than found in M. smegmatis ) and is believed to be involved in the detoxification of the epoxide, isoprene monoxide, formed during the detoxification of isoprene by Rhodococcus sp. AD45.
  • An example of a related compound is the antibiotic cerulenin, an epoxide that reacts with mycothiol and is a substrate of mycothiol conjugate amidase derived from M. smegmatis , as discussed above.
  • bacteria used (or specifically engineered) to detoxify environmental toxins can be transformed with the subject gene constructs to cause or increase expression of acyl glucosaminyl inositol amidase in the bacteria, thereby increasing the capacity of the bacteria to detoxify environmental toxins or expanding the range of toxins against which the bacteria are effective.
  • amidase for substrate was assessed by measuring the production of GlcN-Ins in most cases.
  • a sample (5 ⁇ L) of 1 mM substrate was mixed with 40 ⁇ L of 3 mM 2-mercaptoethanol, 25 mM HEPES chloride, pH 7.5.
  • the reaction was initiated with 5 ⁇ L of purified amidase (50-fold diluted stock, 4.4 ⁇ g ml 31 1 ).
  • Triplicate samples were quenched at 0, 10, and 30 min by mixing each sample with 50 ⁇ L of acetonitrile containing 5 mM NEM and incubating at 60 C for 10 min. After cooling on ice, the samples were clarified by centrifugation for 15 min at 14000 g.
  • a sample (15 ⁇ L) of the supernatant was modified with AccQ-Fluor for amine analysis in a total reaction volume of 125 ⁇ L as previously described (S. J. Anderberg, et al. (1998) supra.).
  • mBBr monobromobimane
  • MPB 3-(N-maleimidopropionyl)biocytin
  • MSA iodoacetamide S-conjugate of mycothiol
  • MSC cerulenin S-conjugate of mycothiol
  • MSH mycothiol, 1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy- ⁇ -D-glucopyranoside
  • MSME NEM S-conjugate of mycothiol
  • MSMC CPM S-conjugate of mycothiol
  • MSMPB MPB S-conjugate of mycothiol
  • MSsB sBBr S-conjugate of mycothiol
  • MSqB qBBr S-conjugate of mycothiol
  • NEM N-ethylmaleimide
  • qBBr monobromotrimethylammoniobimane
  • GlcN-Ins Salt free, stereochemically pure GlcN-Ins was prepared from MSmB by enzymatic hydrolysis using partially purified amidase. A sample of MSmB purified by HPLC (4.8 moles) was incubated in 2 mL of water without buffer at 23° C. with 15 ⁇ g of partially purified amidase. The reaction was monitored hourly for its content of MSmB and AcCySmB and the pH was adjusted to 7.5 with 1M NaOH as necessary.
  • the iced culture was incubated with mBBr (0.5 mM from a 180 mM stock solution in acetonitrile) for 20 min; excess 2-mercaptoethanol (1.0 mM) was added, and the incubation continued on ice for an additional 10 min.
  • the cells were pelleted by centrifugation and washed twice with 200 mL of sterile, ice-cold 7H9 Middlebrook medium to remove excess bimane derivative of 2-mercaptoethanol.
  • the first sample was extracted using 1 mL of 60° C. acetonitrile-water for determination of cellular thiol-bimane derivatives.
  • the second sample was extracted using 1 mL of 60° C. acetonitrile-water containing 2 mM mBBr and 20 mM tris(hydroxymethyl)aminomethane (Tris) pH 8.0 for determination of the sum of each cellular thiol and thiol-bimane derivative.
  • the third sample was extracted using 1 mL of 60° C. acetonitrile-water containing 5 mM N-ethylmaleimide (NEM) and 10 mM HEPES chloride pH 7.5. All tubes were centrifuged at 14000 g in a microcentrifuge and the supernatants removed for analysis.
  • NEM N-ethylmaleimide
  • M. smegmatis cells were cultured as above to late log phase, collected by centrifugation at 5000 g, and frozen at ⁇ 70 C until used.
  • Thawed cell paste (100 gm) was mixed with 500 mL of 3 mM 2-mercaptoethanol, 25 mM HEPES chloride, pH 7.5 (assay buffer) without protease inhibitors and disrupted by sonication on ice.
  • the extract was centrifuged for 30 min at 1 5000 g at 4 C and the supernatant was mixed with saturated ammonium sulfate to 20% saturation and incubated for 1 h on ice.
  • the dialyzed sample was applied to a 1.4 ⁇ 27 cm column of Toyopearl DEAE 650C (TosoHaas) and the column was washed with ⁇ 3 column volumes of assay buffer at 4 C.
  • the column was developed with a linear gradient in assay buffer from 0 to 0.4 M NaCl and the amidase activity eluted at ⁇ 0.2 M NaCl.
  • the active fractions were pooled and saturated ammonium sulfate was added to 20% saturation. After 1 h on ice the solution was clarified by centrifugation for 30 min at 10000 g and the pellet was discarded.
  • the supernatant was applied to a 1.4 ⁇ 27 cm column of Phenyl Sepharose 4B (Sigma) equilibrated with 20% saturated ammonium sulfate in assay buffer at 4° C.
  • the column was washed with 5 column volumes 20% saturated ammonium sulfate followed by 5 column volumes of 10% saturated ammonium sulfate, both in assay buffer.
  • the Phenyl Sepharose 4B column was eluted in assay buffer with a linear gradient from 10% to 0% saturated ammonium sulfate and the amidase activity eluted at ⁇ 1-2% saturated ammonium sulfate.
  • the active fractions were pooled and concentrated at 4 C using a Biomax-50 (Millipore) ultrafilter.
  • the concentrated activity pool was applied to a Sephadex G-100 (Pharmacia) column (1.8 ⁇ 88 cm) equilibrated with assay buffer. The majority of the activity eluted at an estimated M r of 36 000. The most active peak fractions were pooled and concentrated on Centricon C-30 (Amicon) ultrafilters at 4 C. Purified amidase was stored in assay buffer containing 20% glycerol at ⁇ 70° C. for at least 12 months without significant loss of activity.
  • the third step in the three step chromatography of the 20-50% saturated ammonium sulfate fraction had specific activities of ⁇ 3,000 nmol/min-mg protein with 30 ⁇ M MSmB as substrate and were pooled to provide a pure amidase preparation.
  • This sample produced a single band on SDS gel electrophoresis. Only the peak fractions of activity from the gel filtration step were selected and this was the principle factor in reducing the overall yield to 11% as shown in Table 1 above.
  • the amino-terminal sequence of the purified M. smegmatis amidase was determined on an Applied Biosystems Procise Model 494 gas phase protein sequencer by the UCSD Department of Biology Protein Sequencing Facility. Sequencing of the amino-terminal portion of purified amidase produced an amino-terminal sequence of (M)SELRLMAVHAHPDDESSKG (SEQ ID NO:2). The first amino acid was not uniquely defined and its assignment was uncertain until later verified as methionine.
  • a BLAST search (Sanger Centre) of the M. tuberculosis H37Rv genome database (S. T. Cole, et al.
  • Mycothiol S-conjugates were prepared by reaction of excess electrophile with mycothiol followed by removal or reaction of excess electrophile.
  • Stock solutions (100 mM) of electrophile were prepared in acetonitrile (NEM, iodoacetamide; Sigma) or in dimethylsulfoxide [7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM, Molecular Probes), 3-(N-maleimidopropionyl)biocytin (MPB, Sigma)].
  • the purified amidase or dialyzed M. smegmatis extract was incubated in 100 ⁇ M Cysteine, 50 ⁇ M GlcN-Ins, 1 mM ATP, 5 mM MgCl 2 in 50 mM HEPES chloride pH 7.5 at 30° C. and assayed for the time dependent formation of 1-D-myo-inosityl-2-(L-cysteinyl)amido-2-deoxy- ⁇ -D-glucopyranoside (Cys-GlcN-Ins) by HPLC (S. J. Anderberg, et al. (1998) supra.). The reaction was initiated with the addition of the purified amidase (0.044 ⁇ g) or cell extract (50 ⁇ g protein) and was sampled at 0 and 60 min.
  • mycothiol S-conjugate amidase does not appear to be involved in mycothiol biosynthesis since it has no significant ability to catalyze ATP-dependent ligation of cysteine with GlcN-Ins. It therefore does not appear to be a bifunctional enzyme analogous to the glutathionylspermidine synthetase/amidase which catalyzes both the biosynthesis and degradation of glutathionylspermidine in E. coli (D. S. Kwon, et al.(1997) supra) and in Crithidia fasciculata (E. Tetaud, et al. (1998) supra.).
  • M. tuberculosis H37Rv NCTC 7416 was obtained from the National Collection of Type Cultures, London, United Kingdom.
  • E. coli DH5 ⁇ (Clontech Laboratories, Inc., Palo Alto, Calif.) and E. coli BL21(DE3) (Novagen, R & D) were used for maintenance of plasmids and expression of foreign proteins, respectively.
  • the plasmid pET-22b (Novagen) was used as an expression vector in E. coli BL21 (DE3).
  • E. coli strains were cultured on Luria-Bertani (LB) agar or broth with or without selective antibiotics. Mycobacterial strains were cultured in Middlebrook 7H9 broth or 7H10 agar (Difco) supplemented with OADC (Difco) Tween 80, and glycerol.
  • Rv1082 Genomic DNA of M. tuberculosis H37Rv was prepared as described previously (Newton, et al. (1999), supra.).
  • Primers 1 and 2 contained NcoI and BamHI restriction sites respectively.
  • PCR was performed with Taq polymerase obtained from Gibco Brl, using 2 mM MgCl 2 and 5% Dimethyl sulfoxide (DMSO). Annealing temperatures was 50° C. The PCR products were separated on a 1% agarose gel. The appropriate PCR product was ligated into the vector pCR2.1 of the TA cloning kit (Invitrogen) and transformed into E. coli DH5 ⁇ or INVF′ ⁇ by standard chemical transformation procedure. Clones containing the vector were selected on LB+ampicillin (100 ⁇ g/ml) plates and plasmid DNA was digested with restriction endonucleases NcoI and BamHI (Fermentas).
  • Restriction enzyme-digested plasmids were isolated with a QIAquick gel extraction kit (Qiagen Ltd.). A corresponding digestion was also applied to plasmid pET-22b and the two products were ligated together with T4 DNA ligase to obtain the plasmid pYA1082E (FIG. 2).
  • Competent cells of E. coli BL21 were prepared according by the CaCl 2 method (30) and were transformed by the heat shock method for 2 min at 42° C. with 100 ng of pYA1082E (FIG. 2).
  • the transformed E. coli were then plated onto LB agar supplemented with ampicillin (100 ⁇ g/ml). Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 ⁇ g/ml). After over-night incubation at 37° C. with shaking, the individual cultures were diluted 1:50 in the same medium and incubation was continued at 37° C. with shaking.
  • IPTG Isopropyl- ⁇ -D-thiogalacto-pyranoside
  • Amidase inclusion bodies contained in the insoluble fractions were purified from E. coli membrane proteins by centrifugation (45,000 ⁇ g, 90 minutes, 4° C.).
  • the invention amidase was separated by 7.5% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and stained with Coomassie blue. N-terminal amino acid sequence was verified after electrophoresis of samples in SDS-PAGE and electroblotting to PVDF membrane. Edman degradation was performed and the sequence of the first five amino acids from the NH 2 -terminus was determined at the UBC Protein Sequencing Laboratory.
  • Rv 1082 amidase inclusion bodies were resuspended into 1 ⁇ PBS (pH 7.4) and slowly added drop-wise to solution of 16 M Urea and 2 M DTT to make a final concentration of 8 M Urea and 1 M DTT. Soluble amidase was then dialyzed via a Spectra/P or 8000 cellulose membrane (VWR Scientific) against 200 volumes of 1 ⁇ TBS (pH 7.4) at 4° C. for 16-24 hours. The sample was then centrifuged for 15 minutes, 4° C. ⁇ 15,000 g.
  • M. tuberculosis Rv1082 Cloning and expression of the M. tuberculosis Rv1082 gene.
  • the M. tuberculosis Rv1082 gene was cloned and expressed under the control of a T7 promoter in the E. coli expression vector pET-22b.
  • Rv1082 open reading frame was successfully amplified by PCR from M. tuberculosis H37Rv genomic DNA to give a 858 bp fragment and cloned into the T7 expression vector pET-22b.
  • the map of the resulting plasmid described as pYA1082E is shown in FIG. 2.
  • tuberculosis was expressed from pYA1082E following treatment of exponentially growing pYA1082E- E. coli BL21 (DE3) transformed cells with 1 mM IPTG at room temperature for about 12 hours .
  • IPTG induced a protein approximately 37 kDa in size. This expressed band was visible in both whole cell lysates and post-sonication pellet within two hours of IPTG induction.
  • Further purification attempts revealed that mycothiol S-conjugate amidase was present in the form of insoluble inclusion bodies. The inclusion bodies remained as stable insoluble aggregates even following multiple washes with detergent solutions.
  • Cells were pelleted by centrifugation for 10 min at 5000 g and 4° C., sonicated in 5 volumes of assay buffer on ice, and centrifuged for 5 min at 14000 g at room temperature. About 85% of the amidase activity, using MSmB as substrate, was associated with the pellet fraction which was resuspended and incubated with periodic vortexing for 1 h at 37° C. in 8 M urea containing 20 mM DTT.
  • the suspension was centrifuged for 3 min at 14000 g and the supernatant dialyzed against 100 volumes of 25 mM HEPES chloride pH 7.5 containing 1 mM glutathione disulfide and 2 mM glutathione for 15 h, and then against 100 volumes of 25 mM HEPES chloride pH 7.5 containing 3 mM 2-mercaptoethanol for 4 h. After centrifugation, the supernatant contained soluble amidase activity and was assayed with 0.1 mM mycothiol and 0.1 mM MSmB as described above.
  • M. tuberculosis mycothiol S-conjugate amidase possesses functional mycothiol-S-conjugate amide hydrolase activity. Alkylation of MSH with mBBr produces the fluorescent S-conjugate, MSmB, which can be quantitated by HPLC with fluorescence detection (Newton et al. 1995; Newton et al. 1996). E coli has no mycothiol metabolism and is not expected to contain mycothiol conjugate amidase endogenous proteins that would give background to these assays. The amidase activity was found to be associated with the insoluble cell pellet material.
  • the resolublilzed crude protein extract was found to produce 4.1 ⁇ 0.05 nmoles/min/mg protein GlcN-Ins and 5.4 ⁇ 0.3 nmoles/min/mg protein AcCysmB.
  • the S-conjugate amidase which is encoded by the M. tuberculosis open reading frame Rv1082 is 288 amino acid long, slightly negatively charged peptide with a predicted molecular weight of 32699 da and theoretical PI of 5.11.
  • thiol analysis of Rhodococcus sp. Strain AD45 was conducted.
  • the thiol analysis shows that mycothiol is the major thiol, with glutathione as a minor thiol at about 10% of the mycothiol level.
  • Further analysis of the mycothiol S-conjugate amidase from this bacterium showed amidase activity with mycothiol-bimane derivative as substrate was 2-fold higher than that found in Mycobacterium smegmatis (Table 3).
  • the high mycothiol content relative to the glutathione content of this organism along with the inability to saturate the glutathione S-transferase with the substrate glutathione suggests that the active amidase in this organism is actually a mycothiol S-transferase and not a glutathione S-transferase.
  • the mycothiol S-conjugate amidase present in this organism (at levels higher than found in M. smegmatis ) (Table 3) is believed to be involved in the detoxification of the epoxide, isoprene monoxide, formed during the detoxification of isoprene by Rhodococcus sp. AD45.
  • An example of a related compound is the antibiotic cerulenin, an epoxide that reacts with mycothiol and is a substrate of mycothiol conjugate amidase from M. smegmatis .
  • Table 3 shows the thiol content and mycothiol S-conjugate amidase in crude cell extracts of Rhodococcus sp. AD45 and Mycobacterium smegmatis mc 2 155. TABLE 3 Amidase Thiol Content ⁇ Moles/gram activity residual dry weight Units/mg Organism Cysteine Glutathione Mycothiol protein Rhodococcus sp. 0.31 1.1 12.6 0.0025 AD45 M. smegmatis 0.16 ⁇ 0.001 10.6 0.0012 mc 2 155 a
  • mycothiol is the major low molecular weight thiol and will form a mycothiol conjugate.
  • the product of this conjugation may still be toxic and is a substrate for the mycothiol conjugate amidase.
  • Reaction of the mycothiol conjugate with a mycothiol conjugate amidase enables the excretion of the detoxified conjugate as a mercapturic acid. Therefore, mycothiol and mycothiol S-conjugate amidase are involved in and can be used for detoxification of halogenated hydrocarbons and other environmental toxins.
  • bacteria transformed with a polynucleotide encoding an invention amidase polypeptide, or a variant thereof is used to express the amidase in situ under environmental conditions and the toxic substance is an environmental pollutant, such as a halogenated hydrocarbon, hydrocarbon, or other petroleum derivative.

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