WO2022168952A1

WO2022168952A1 - Novel prenylation enzyme

Info

Publication number: WO2022168952A1
Application number: PCT/JP2022/004501
Authority: WO
Inventors: 敏幸脇本; 龍文沖野; 研一松田; チンスンパン
Original assignee: 国立大学法人北海道大学
Priority date: 2021-02-05
Filing date: 2022-02-04
Publication date: 2022-08-11
Also published as: JPWO2022168952A1

Abstract

The present invention provides: an enzyme that transfers a prenyl group to an arginine residue or an arginine analog residue in a peptide, or to arginine or an arginine analog, wherein the enzyme includes an amino acid sequence represented by SEQ ID NO: 1 or a mutant sequence thereof; a peptide that can be used as a substrate for the enzyme; and a prenylated peptide.

Description

Novel prenylating enzyme

The present invention relates to prenylation-modifying enzymes for peptides and amino acids, substrates for the enzymes, and the like. Specifically, the present invention relates to an enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue in a peptide, or to an arginine or an arginine analogue, and a peptide that can be used as a substrate for the enzyme.

Cyanobactins, peptide compounds produced by blue-green algae, often show regioselective prenylation modification. A plurality of prenyltransferases have been discovered so far (Patent Document 1, Non-Patent Documents 1 to 3), and all of them exhibit high regioselectivity and tolerant substrate selectivity in vitro. Since these act on cyclic peptides, they are useful as diversity creation tools in the final stage of synthesis. However, the prenylation sites of these enzymes are limited to the side chains of tryptophan, tyrosine, serine, and threonine residues and the N- and C-termini of linear peptides, which creates limited structural diversity. .

United States Patent Application Publication US2010/0209414A1

It is necessary to create peptides with diverse structures and functions. For that purpose, it is necessary to find an enzyme that has properties different from those of conventional prenyltransferases. There is also a need to find peptides and amino acids that serve as substrates for such enzymes.

As a result of intensive research to solve the above problems, the present inventors have found a novel enzyme that transfers a prenyl group to an arginine residue in a peptide and a peptide that can be used as a substrate for this enzyme in cyanobacteria. , have completed the present invention.

Accordingly, the present invention provides:
(1) An enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue, or an arginine or an arginine analogue in a peptide containing the following amino acid sequence:
(a) the amino acid sequence of SEQ ID NO: 1;
(b) an amino acid sequence having 58% or more identity to the amino acid sequence of SEQ ID NO: 1, or (c) deletion or substitution of 1 to several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 , inserted or added amino acid sequence.
(2) a nucleic acid comprising the following nucleotide sequence:
(a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1;
(b) a nucleotide sequence encoding an amino acid sequence having 58% or more identity to the amino acid sequence of SEQ ID NO: 1;
(c) a nucleotide sequence encoding an amino acid sequence in which one to several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1, or (d) the amino acid shown in SEQ ID NO: 1 A base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence encoding the sequence.
(3) An enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue in a peptide, or an arginine or an arginine analogue encoded by the nucleic acid of (2).
(4) A vector comprising the nucleic acid according to (2).
(5) A method for producing an enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue in a peptide, or an arginine or an arginine analogue, comprising the following steps:
(a) introducing the nucleic acid according to (2) or the vector according to (4) into a cell;
(b) culturing the cells obtained in step (a); and (c) arginine residues or arginine analogue residues, or arginine or arginine analogues in peptides from the culture obtained in step (b). Obtaining an enzyme that prenylates the body.
(6) the following (i) and (ii):
(i) a peptide containing an arginine residue or an arginine analogue residue, or an arginine or an arginine analogue (ii) an arginine residue comprising allowing the enzyme of (1) or (3) to act on a prenyl group-containing compound A method for producing a peptide having a prenylated group or arginine analogue residue, or a prenylated arginine or arginine analogue.
(7) The method according to (6), wherein the peptide is a cyclic peptide.
(8) in the peptide, a proline residue is adjacent to an arginine residue or an arginine analogue residue, and the proline residue is bound via its carboxyl group to the amino group of the arginine residue or the arginine analogue residue; (6) or (7).
(9) The enzyme contains an amino acid sequence in which the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than histidine, or the amino acid sequence shown in SEQ ID NO: 1 Any one of (6) to (8), which comprises an amino acid sequence in which the amino acid residue corresponding to the 167th amino acid residue from the N-terminus is an amino acid residue other than histidine, and the prenylation is monoprenylation The method described in .
(10) The method according to item (9), wherein the amino acid other than histidine is alanine.
(11) an arginine residue or an arginine analogue, comprising allowing the enzyme of

claim

1 or 3 to act on a peptide containing an arginine residue or an arginine analogue residue, or a compound library containing arginine or an arginine analogue; A method for producing a compound library containing peptides prenylated at the amino acid residue, or prenylated arginines or arginine analogues.
(12) The method according to (11), wherein the peptide is a cyclic peptide.
(13) in the peptide, a proline residue is adjacent to an arginine residue or an arginine analogue residue, and the proline residue is bound to the amino group of the arginine residue or the arginine analogue residue through its carboxyl group; (11) or (12).
(14) The enzyme contains an amino acid sequence in which the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than histidine, or the amino acid sequence shown in SEQ ID NO: 1 Any one of (11) to (13), which comprises an amino acid sequence in which the amino acid residue corresponding to the 167th amino acid residue from the N-terminus is an amino acid residue other than histidine, and the prenylation is monoprenylation The method described in .
(15) The method according to item (14), wherein the amino acid other than histidine is alanine.
(16) Formula (I):

(I)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
A compound represented by
(17) Formula (II):

(II)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
A compound represented by
(18) Formula (III):

(III)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
A compound represented by
(19) Formula (IV):

(IV)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
A compound represented by

According to the present invention, there is provided an enzyme that transfers a prenyl group to an arginine residue or arginine analogue residue in a peptide, or to arginine or an arginine analogue. The enzymes can be used to create peptides and amino acids with diverse structures and functions. Furthermore, the present invention provides peptides that can be used as substrates for the enzyme. The enzyme can act on various peptides and amino acids to create new prenylated peptides and prenylated amino acids with diverse structures and functions.

Figure 1 shows LC-MS charts of algicyclamide C (3) isolated from Microcystis aeruginosa NIE-88 (upper), chemically synthesized algicyclamide C (3) (middle), and a mixture of both (lower). It is a figure which compared. FIG. 2 is a diagram comparing ¹ H NMR charts of Argicyclamide C (3) isolated from Microcystis aeruginosa NIE-88 (upper) and chemically synthesized Argicyclamide C (3) (lower). FIG. 3 is a diagram comparing ¹³ C NMR charts of Argicyclamide C (3) isolated from Microcystis aeruginosa NIE-88 (upper) and chemically synthesized Argicyclamide C (3) (lower). FIG. 4 is a reverse-phase HPLC chart showing the time-dependent changes (0 to 120 minutes) of substrates and products in the conversion reaction of algicyclamide C (3) to algicyclamide A (1) by the enzyme (AgcF) of the present invention. is. Boiled AgcF is a chart of the reaction product using the enzyme of the present invention heat-inactivated before the reaction. 1 (std) is a chart of algicyclamide A (1) standard, 2 (std) is a chart of algicyclamide B (2) standard, and 3 (std) is a chart of algicyclamide C (3) standard. The right panel of FIG. 5 shows the ratio of monoprenylated and bisprenylated forms produced by prenylation reaction by allowing AgcF to act on a cyclic peptide in which each amino acid residue contained in the sequence of algicyclamide C is substituted with alanine. shows the results of examining The scheme in the figure shows the progress of the prenylation reaction of Argicyclamide C with AgcF. FIG. 6 shows an HPLC chart of the monoprenylation reaction of Argicyclamide C by AgcF_H167A. The scheme in the figure shows the monoprenylation reaction of Argicyclamide C by AgcF_H167A. FIG. 7A shows the UPLC chromatogram of the prenylation reaction of the substrate _cyc [MEYPLSLRYPG] by the AgcF homolog recombinant UHCC0183PT (monitoring UV absorbance at 210 nm). i indicates a chromatogram of a reaction solution containing recombinant UHCC0183PT. ii is the chromatogram of the reaction without recombinant UHCC0183PT. FIG. 7B shows the results of UPLC-MS analysis (positive mode) of the reaction mixture containing UHCC0183PT. i indicates the extracted ion chromatogram at m/z 1443.0 corresponding to the bisprenylated form. ii shows the extracted ion chromatogram at m/z 1375.0 corresponding to the monoprenylated form. iii shows the extracted ion chromatogram at m/z 1307.0 corresponding to the substrate.

The terms used in this specification have meanings commonly understood in the fields of biology, biochemistry, chemistry, pharmacy, medicine, etc., unless otherwise specified. Amino acid designations herein are based on the known one-letter or three-letter system.

In one aspect, the present invention provides an enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue, or an arginine or an arginine analogue, in a peptide. The enzyme is
(a) the amino acid sequence of SEQ ID NO: 1;
(b) an amino acid sequence having 58% or more identity to the amino acid sequence of SEQ ID NO: 1, or (c) deletion or substitution of 1 to several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 , including inserted or added amino acid sequences.

The enzyme of the present invention uses peptides containing arginine or arginine analogues, or arginine or arginine analogues (prenyl group acceptors) and prenyl group-containing compounds (prenyl group donors) as substrates, and arginine residues in peptides or It catalyzes the transfer of a prenyl group to an arginine analogue residue, or to arginine or an arginine analogue. Specifically, the enzyme of the present invention transfers a prenyl group to the side chain nitrogen atom of an arginine residue or arginine analogue residue in a peptide, or to the nitrogen atom of arginine or an arginine analogue (however, the above nitrogen atom does not constitute a peptide bond). When the prenyl group acceptor contains multiple nitrogen atoms to which a prenyl group can be bonded, the prenyl group may be transferred to one of them, or the prenyl group may be transferred to two or more of them. For example, in the prenyl group transfer reaction by the enzyme of the present invention using a peptide containing an arginine residue as a prenyl group acceptor, a prenyl group may be bound to one nitrogen of the guanidine of the arginine residue, and two prenyl groups may be bound to the two nitrogens. groups may be attached.

The structure of the prenyl group acceptor peptide is not particularly limited, and may be linear, branched or cyclic. Also, for example, the peptide may be a peptide in which a linear peptide and a cyclic peptide are bound. Preferably the peptide is a cyclic peptide. Peptides may be naturally occurring peptides or artificially created peptides. The size of the peptide is not particularly limited, and may consist of, for example, several amino acids, several tens of amino acids, or a larger size. As used herein, a peptide may be an oligopeptide, polypeptide or protein. The enzymes of the invention can be used to prenylate oligopeptides, polypeptides and proteins.

The arginine residue or arginine analogue residue may be present at any position in the peptide. Arginine residues or arginine analogue residues may be present at the N-terminus, C-terminus, or both of the peptide, and may be internal to the peptide.

In the peptide, the proline residue is preferably adjacent to an arginine residue or an arginine analogue residue. Preferably, a proline residue is adjacent to an arginine residue or arginine analogue residue by binding the proline residue through its carboxyl group to the amino group of the arginine residue or arginine analogue residue. Preferred peptides for prenylation by the enzymes of the invention are cyclic peptides in which proline residues are flanked by arginine or arginine analogue residues in the manner described above.

Amino acids that constitute peptides are usually amino acids that constitute proteins and are in the L-form, but they may be amino acids that do not constitute proteins, or may be in the D-form. For example, the amino acids that make up the peptide may be β-alanine, γ-aminobutyric acid, ε-aminocaproic acid, etc., and may also be alkylated, halogenated, esterified, aminated, carboxylated, nitrated, sulfonated, etc. It may be an amino acid that has been modified with a modified, phosphorylated, acetylated, glycosylated, lipidated, etc., or known protective group. Peptides containing these modified amino acids can be obtained by known methods.

Amino acid bonds in peptides are usually peptide bonds, but may contain other bonds such as ester bonds, and may have spacers such as methylene groups between amino acid residues.

The enzyme of the present invention can also use arginine or an arginine analogue as a prenyl group acceptor. As used herein, arginine analogues include derivatives of arginine, amino acids having similar structures to arginine, and derivatives thereof. Here, derivatives include modifications such as alkylation, halogenation, esterification, amination, carboxylation, nitration, sulfonation, phosphorylation, acetylation, glycosylation, lipidation, etc. It may be a compound. Moreover, the derivative may be one to which a known protecting group is attached. Derivatives are not limited to the above examples. Arginine analogs can be obtained by known methods. Representative examples of analogs of arginine include, but are not limited to, ornithine, citrulline, and argininosuccinic acid, and derivatives thereof. The above comments also apply to analogue residues of arginine in peptides.

The prenyl group transferred by the enzyme of the present invention may be any prenyl group, such as dimethylallyl, geranyl, farnesyl, geranylgeranyl, geranylfarnesyl, hexaprenyl, octaprenyl, decaprenyl. etc., but not limited to these. The size of the prenyl group transferred by the enzyme of the present invention preferably has 20 carbon atoms or less, more preferably 15 carbon atoms or less.

The prenyl group-containing compound (prenyl group donor) is not particularly limited, and may be any type of compound. Examples of prenyl group-containing compounds include, but are not limited to, dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and the like.

The enzyme of the present invention is a novel enzyme in that it transfers a prenyl group to an arginine residue or arginine analogue residue in a peptide, or to arginine or an arginine analogue. The enzyme of the present invention is useful as a peptide modification tool for creating new peptides with diverse structures and functions that have been impossible with conventional enzymes.

The enzyme of the present invention may be derived from any organism. The enzymes of the invention are preferably of bacterial, more preferably cyanobacterial origin. Enzymes of the present invention may be derived from cyanobacteria such as, for example, the genera Microcystis, Aphanizomenon, and Dolichospermum. The enzyme of the invention may be, for example, one comprising the amino acid sequence shown in SEQ ID NO: 1 (AgcF).

The enzyme of the present invention can include its mutants. In the present specification, unless otherwise specified, the enzyme of the present invention includes the enzyme of the present invention and variants thereof.

A mutant of the enzyme of the present invention may contain a mutant sequence of the amino acid sequence shown in SEQ ID NO: 1.

For example, the mutant of the enzyme of the present invention is 50% or more, for example, 58% or more, 60% or more, 70% or more, 80% or more, 90% or more, 92% or more of the amino acid sequence shown in SEQ ID NO: 1 , may include amino acid sequences with greater than 94%, greater than 96% or greater than 98% identity. The identity of amino acid sequences can be examined using known means such as FASTA search and BLAST search.

Further, for example, the mutant of the enzyme of the present invention has one to several tens, preferably one to several amino acid residues deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1. May contain sequences. The number of amino acid residues to be deleted, substituted, inserted or added in SEQ ID NO: 1 is not limited to 1 to several, but 1 to several tens, preferably 1 to 40, more preferably may be 1 to 20, more preferably 1 to several. Dozens may be, for example, 20, 30, 40, 50, 60, 70, 80, 90, or any number between these values. Several may be, for example, 2, 3, 4, 5, 6, 7, 8 or 9. Deletions, substitutions, insertions or additions of amino acid residues in protein amino acid sequences are known to those skilled in the art. For example, site-directed mutagenesis or known chemical techniques may be used to create deletions, substitutions, insertions or additions of amino acid residues in the amino acid sequences of the enzymes of the invention. In the case of substitution of amino acid residues, substitutions between homologous amino acids are preferred. Cognate amino acids are known to those of skill in the art. Conservative amino acid substitutions are also preferred. Examples of conservative amino acid substitutions include: substitutions between Phe, Trp, Tyr when the substituted amino acid is an aromatic amino acid, when the substituted amino acid is a hydrophobic amino acid for substitutions between Leu, Ile, and Val; substitutions between Gln and Asn when the amino acid to be substituted is a polar amino acid; Lys when the amino acid to be substituted is a basic amino acid; Substitution between Arg and His, substitution between Asp and Glu when the amino acid to be substituted is an acidic amino acid, substitution between Ser and Thr when the amino acid to be substituted is an amino acid having a hydroxyl group replacement.

Preferred examples of mutants of the enzyme of the present invention include amino acid sequences shown in SEQ ID NO: 1 from the N-terminus of the 67th glycine, the 133rd glycine, the 219th cysteine, the 267th cysteine, and the 289th leucine. Enzymes are included that contain amino acid sequences in which the corresponding amino acid residues are the same as, their cognate amino acids, or amino acids with conservative amino acid substitutions therefrom. Preferably, the amino acid sequence of such a mutant enzyme is 50% or more, for example, 58% or more, 60% or more, 70% or more, 80% or more, 90% or more of the amino acid sequence shown in SEQ ID NO: 1, 92% or greater, 94% or greater, 96% or greater or 98% or greater identity. Examples of such preferred variants of the enzyme of the present invention include the 67th glycine, 133rd glycine, 219th cysteine, 267th cysteine and 289th cysteine from the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1. The amino acid residue corresponding to the th leucine is glycine, glycine or alanine, cysteine, cysteine, and leucine, respectively, and 58% or more, 60% or more, 70% or more, and 80% of the amino acid sequence shown in SEQ ID NO: 1 % or greater, 90% or greater, 92% or greater, 94% or greater, 96% or greater or 98% or greater amino acid sequences, including, but not limited to, such enzymes. The "corresponding amino acid residue" in the amino acid sequence of the mutant enzyme can be found by considering the position counted from the N-terminus and the neighboring amino acid sequences, structural prediction by computer, and the like.

The mutant of the enzyme of the present invention may be naturally derived, or may be artificially produced using, for example, genetic engineering techniques. Variants of the enzymes of the invention may be derived from any organism. The variants of the enzymes of the invention are preferably of bacterial, more preferably cyanobacterial origin. Variants of the enzymes of the invention may be derived from cyanobacteria such as, for example, the genera Microcystis, Aphanizomenon, Dolichospermum.

Examples of variants of the enzyme of the present invention include, but are not limited to, AgcF homologues UHCC0183PT and PCC9443PT. The amino acid sequence of UHCC0183PT is shown in SEQ ID NO:6, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO:7. The amino acid sequence of PCC9443PT is shown in SEQ ID NO:9, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO:10. The amino acid sequence of UHCC0183PT (SEQ ID NO:6) has 59.86% identity to the amino acid sequence of AgcF (SEQ ID NO:1). The amino acid sequence of PCC9443PT (SEQ ID NO:9) has 58.54% identity to the amino acid sequence of AgcF (SEQ ID NO:1).

The enzyme of the present invention can be obtained using a known method. For example, AgcF may be obtained by cloning the AgcF gene using PCR, ligating it into an expression vector, introducing the expression vector into host cells, and culturing the host cells. Mutations of the enzymes of the invention can also be obtained using known methods. For example, AgcF variants may be obtained by cloning AgcF homologues or orthologs, ligating into an expression vector, introducing the expression vector into host cells, and culturing the host cells. An AgcF homolog may be obtained by synthesizing a gene with reference to the published sequence of the AgcF homolog, introducing it into a host cell, and culturing the host cell. AgcF mutants can also be obtained by modifying the base sequence of the gene encoding AgcF using a known method such as site-directed mutagenesis, introducing the modified gene into host cells, and culturing the host cells. good. Mutants of the enzyme of the present invention may be obtained by chemical methods such as chemical synthesis and chemical modification.

Alternatively, the mutant of the enzyme of the present invention may contain non-proteinogenic amino acids, D-form amino acids, modified amino acids, and the like.

30% or more, preferably 50% or more, more preferably 70% or more, even more preferably 80% or more, most preferably 80% or more, of the enzyme of the invention comprising the amino acid sequence shown in SEQ ID NO: 1 has a prenyl group transfer activity of 90% or more.

An enzyme comprising an amino acid sequence in which the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is a histidine residue, or an enzyme containing an amino acid sequence at the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 Bisprenylation of arginine or arginine analogue residues, or arginine or arginine analogues in peptides is enhanced when enzymes containing amino acid sequences in which the corresponding amino acid residue is a histidine residue are used.

An enzyme comprising an amino acid sequence in which the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than histidine, or the 167th amino acid from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 Bisprenylation of an arginine residue or an arginine analogue residue in a peptide is inhibited and monoprenylation is inhibited when an enzyme containing an amino acid sequence in which the amino acid residue corresponding to the residue is an amino acid residue other than histidine is used. Promoted. Examples of amino acid residues other than histidine include, but are not limited to, alanine residues.　

The prenyl group transfer activity can be measured by a known method. For example, the enzyme of the present invention is added to a reaction solution containing a peptide containing an arginine residue and a compound containing a prenyl group, and the unit amount of the enzyme and the amount of prenylated peptide produced per unit time are determined by the prenylation of the enzyme of the present invention. It may also be used as an indicator of group transfer activity. Confirmation of production of the prenylated peptide and measurement of the production amount can be performed using known means and methods such as NMR, MS, HPLC and LC-MS.

In another aspect, the present invention provides the following nucleotide sequence:
(a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1;
(b) a nucleotide sequence encoding an amino acid sequence having 58% or more identity to the amino acid sequence of SEQ ID NO: 1;
(c) a nucleotide sequence encoding an amino acid sequence in which one to several tens, preferably one to several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1, or (d ) Providing a nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1.

The identity of the amino acid sequences is as explained above. The number of amino acid residues to be deleted, substituted, inserted or added is also as described above.

Stringent conditions are known to those of skill in the art and include, for example:
in a buffer containing 0.25 M Na ₂ HPO ₄ , pH 7.2, 7% SDS, 1 mM EDTA, 1× Denhardt's solution at a temperature of 60 to 68° C., preferably 65° C., more preferably 68° C. Hybridize for 16 to 24 hours, then in a buffer containing 20 mM Na ₂ HPO ₄ , pH 7.2, 1% SDS, 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, more preferably 68°C. conditions with two 15 minute washes under
42 in a hybridization solution containing 25% formamide, or even 50% formamide, 4×SSC (sodium chloride/sodium citrate), 50 mM HEPES pH 7.0, 10× Denhardt's solution, 20 μg/ml denatured salmon sperm DNA. After overnight prehybridization at °C, in a buffer containing 1 x SSC, 0.1% SDS at 37 °C, or more stringent conditions, a buffer containing 0.5 x SSC, 0.1% SDS. 0.2 x SSC, 0.1% as a more severe condition at 42 ° C in liquid
Conditions for washing at 65° C. in buffer containing SDS.
Needless to say, stringent conditions are not limited to the above examples.

As used herein, nucleic acids can include the DNAs described, DNAs complementary to the DNAs, and RNAs complementary to these DNAs.

As used herein, a nucleotide sequence includes a degenerate sequence that encodes the desired amino acid sequence.

The present invention, in another aspect,
(e) the nucleotide sequence shown in SEQ ID NO: 2, or (f) a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2, to provide a nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions. .

The base sequence (e) above is an example of the base sequence (a) that encodes the amino acid sequence shown in SEQ ID NO: 1. The nucleotide sequence (f) above is an example of a nucleotide sequence (d) that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1.

The stringent conditions and nucleic acids are as described above.

The above nucleic acids can be obtained by methods known to those skilled in the art. Said nucleic acid may be obtained by cloning the gene encoding the enzyme of the invention or its homologue or ortholog. Cloning techniques are known to those of skill in the art. The source of the nucleic acid is not particularly limited, but is preferably bacteria, more preferably blue-green algae. Examples of the cyanobacteria from which the nucleic acid is derived include, but are not limited to, cyanobacteria belonging to the genera Microcystis, Aphanizomenon, and Dolichospermum. Alternatively, the above nucleic acid may be obtained by artificial synthesis.

In another aspect, the present invention provides an enzyme encoded by the above nucleic acid that transfers a prenyl group to an arginine residue or an arginine analogue residue, or an arginine or arginine analogue residue in a peptide. .

In another aspect, the present invention provides a vector containing the above nucleic acid. An expression vector is preferred as the vector. Various expression vectors are known and can be appropriately selected and used. Methods for incorporating the DNA of the present invention into vectors are also known.

The above nucleic acid encodes the enzyme of the present invention. In addition, the vector contains the nucleic acid. Therefore, the enzyme of the present invention can be produced by genetic engineering using the above nucleic acid or vector.

Accordingly, in another aspect, the present invention provides a method for producing an enzyme that transfers a prenyl group to an arginine residue or arginine analogue residue in a peptide, or arginine or an arginine analogue. The method comprises the following steps:
(a) introducing said nucleic acid or said vector into a cell;
(b) culturing the cells obtained in step (a), and (c) from the culture obtained in step (b), an arginine residue or an arginine analog residue, or arginine or arginine in the peptide Obtaining an enzyme that transfers a prenyl group to an analogue.

For example, the nucleic acid may be incorporated into an expression vector, introduced into cells, and the enzyme of the present invention obtained by culturing the cells. Alternatively, for example, the enzyme of the present invention may be obtained from a culture obtained by introducing the nucleic acid or the vector into cells using a known method such as the PEG method, electroporation, or particle gun method, and culturing the cells. good.

The cells used in the above method are not particularly limited, and may be microbial cells such as bacteria, yeast, filamentous fungi, and actinomycetes, plant cells, animal cells, and insect cells. Preferred cells for use in this method include, but are not limited to, microbial cells such as E. coli and Bacillus subtilis.

Cell culture can be performed by a known method. Selection of cells and culture conditions is routine for those of skill in the art.

In the case of cells that produce the enzyme of the present invention within the cells, the cells are disrupted by known means such as ultrasonic waves, mills, homogenizers, etc. to obtain an extract, and then subjected to known means such as ammonium sulfate precipitation and chromatography. The enzyme of the present invention can be obtained from the extract. In the case of cells that extracellularly produce the enzyme of the present invention, the enzyme of the present invention can be obtained by subjecting the culture medium to known means such as ammonium sulfate precipitation and chromatography.

In addition to the genetic engineering method described above, the enzyme of the present invention may be obtained by extraction and purification from organisms such as bacteria, preferably cyanobacteria of the genus Microcystis, Aphanizomenon, and Dolichospermum. Known enzyme purification means and methods such as cell disruption, ammonium sulfate precipitation, and chromatography can be used.

In a further aspect, the present invention provides the following (i) and (ii):
(i) peptides containing arginine residues or arginine analogue residues, or arginine or arginine analogues (prenyl group receptors)
(ii) prenyl group-containing compound (prenyl group donor)
Provided is a method for producing a peptide in which an arginine residue or an arginine analogue residue is prenylated, or a prenylated arginine or an arginine analogue, comprising allowing the enzyme of the present invention to act on .

Peptides containing arginine residues or arginine analogues, arginine or arginine analogues, prenyl group-containing compounds, and prenyl groups contained in the compounds are as described above.

By adding the enzyme of the present invention to a reaction solution containing a prenyl group acceptor and a prenyl group donor and incubating under appropriate conditions, a peptide in which an arginine residue or an arginine analogue residue is prenylated, or a prenyl modified arginine or arginine analogs can be obtained. Incubation conditions can be appropriately determined by those skilled in the art according to the properties and amount of the enzyme, the type and concentration of the substrate, and the like. Suitable conditions include, for example, room temperature to 37° C., pH 6 to 9, and reaction conditions for several hours, but are not limited to these conditions. The enzyme of the present invention may be immobilized on a carrier and used.

Peptides in which arginine residues or arginine analogue residues are prenylated, or prenylated arginine or arginine analogues can be purified and isolated from the reaction solution using known techniques such as various chromatography. .

The production of these prenylated compounds can be confirmed by known means. For example, confirmation may be performed by analyzing the reaction mixture using high performance liquid chromatography (HPLC). Mass spectroscopy may be used to confirm the molecular weight of the product when the prenyl group acceptor is prenylated. NMR may be used to further determine the structure of the product. Product confirmation is preferably performed by comparison with a standard preparation of the prenylated compound. The preparation of the prenylated compound may be a commercial product, one obtained by chemical synthesis, or one isolated from nature.

In a further aspect, the present invention provides an arginine residue or an arginine analogue comprising allowing the enzyme of the present invention to act on a peptide containing an arginine residue or an arginine analogue residue, or a compound library containing arginine or an arginine analogue. Provided are methods for producing a compound library containing peptides prenylated at the amino acid residue, or prenylated arginines or arginine analogues.

Peptides containing arginine residues or arginine analogue residues, or compound libraries containing arginine or arginine analogues can be obtained by known methods using arginine or arginine analogues as part of the starting material. Such known methods include, but are not limited to, combinatorial-split synthesis methods, peptide array methods, bead display methods, phage display methods, and the like.

When a peptide is used as a prenyl group acceptor in the above method for producing a peptide in which an arginine residue or an arginine analogue residue is prenylated, or a prenylated arginine or an arginine analogue, and a method for producing a compound library is preferably a cyclic peptide.

In the peptide, the proline residue is preferably adjacent to an arginine residue or an arginine analogue residue. Preferably, a proline residue is adjacent to an arginine residue or arginine analogue residue by binding the proline residue through its carboxyl group to the amino group of the arginine residue or arginine analogue residue. In the method for producing a peptide in which an arginine residue or an arginine analogue residue is prenylated, or the method for producing a prenylated arginine or an arginine analogue, and the method for producing a compound library, the preferred peptide is a cyclic peptide, , in which proline residues are flanked by arginine or arginine analog residues in the manner described above.

When it is desired to obtain a large amount of bisprenylated products in the above-described method for producing a peptide in which an arginine residue or an arginine analog residue is prenylated, or a method for producing a prenylated arginine or an arginine analog, and a method for producing a compound library is an enzyme containing an amino acid sequence in which the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is a histidine residue, or the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 It is preferred to use an enzyme comprising an amino acid sequence in which the amino acid residue corresponding to the group is a histidine residue.

When it is desired to obtain a large amount of monoprenylated products in the above-described method for producing a peptide in which an arginine residue or an arginine analogue residue is prenylated, or a method for producing a prenylated arginine or an arginine analogue, and a method for producing a compound library is an enzyme comprising an amino acid sequence in which the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than histidine, or the 167th from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 It is preferred to use an enzyme comprising an amino acid sequence in which the amino acid residue corresponding to the amino acid residue of is an amino acid residue other than histidine. Examples of amino acid residues other than histidine include, but are not limited to, alanine residues. For example, the amino acid residue corresponding to the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 in AgcF homolog UHCC0183PT is the 49th glutamic acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 6. is. Further, for example, the amino acid residue corresponding to the 167th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 in AgcF homolog PCC9443PTT is the 49th glutamic acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 9. is the base.

A method of causing the enzyme of the present invention to act on a peptide containing an arginine residue or an arginine analogue residue, or a compound library containing arginine or an arginine analogue is a peptide in which an arginine residue or an arginine analogue residue is prenylated. Alternatively, it may be according to the method described for the method for producing prenylated arginine or arginine analogues.

By screening the obtained compound library, it is possible to search and obtain prenylated peptides and prenylated amino acids according to the purpose.

The present invention provides, in another aspect, a compound of formula (I):

(I)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
to provide a compound represented by

The compound of formula (I) in which R ₁ and R ₂ are both hydrogen is algicyclamide C (see Examples). Argicyclamide C was isolated and identified from Microcystis aeruginosa NIE-88, which is a blue-green algae, and is a compound that can be used as a substrate for the enzyme of the present invention. This compound may be obtained from Microcystis aeruginosa NIE-88, or may be obtained by a known synthesis method (chemical synthesis method, enzymatic synthesis method, etc.).

A compound of formula (I) in which either one of R ₁ and R ₂ is a prenyl group is a monoprenylated compound. Compounds of formula (I) in which both R ₁ and R ₂ are prenyl groups are bisprenylated.

These monoprenylated forms and bisprenylated forms may be obtained by allowing the enzyme of the present invention to act on the compound represented by formula (I) in which both R ₁ and R ₂ are hydrogen, or may be obtained by a known synthetic method ( chemical synthesis method, enzymatic synthesis method, etc.).

The compound represented by formula (I) was newly isolated, identified and synthesized this time.

The present invention provides, in another aspect, a compound of formula (II):

(II)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
to provide a compound represented by

Compounds of formula (II) in which R ₁ and R ₂ are both hydrogen may be used as substrates for enzymes of the invention.

A compound of formula (II) in which either one of R ₁ and R ₂ is a prenyl group is a monoprenylated compound. Compounds of formula (II) in which both R ₁ and R ₂ are prenyl groups are bisprenylated.

These monoprenylated forms and bisprenylated forms may be obtained by allowing the enzyme of the present invention to act on the compound represented by formula (II) in which both R ₁ and R ₂ are hydrogen, or may be obtained by a known synthetic method ( chemical synthesis method, enzymatic synthesis method, etc.).

The compound represented by formula (II) was newly synthesized and identified this time.

The present invention provides, in another aspect, a compound of formula (III):

(III)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
to provide a compound represented by

Compounds of formula (III) in which R ₁ and R ₂ are both hydrogen may be used as substrates for enzymes of the invention.

A compound of formula (III) in which either one of R ₁ and R ₂ is a prenyl group is a monoprenylated compound. Compounds of formula (III) in which both R ₁ and R ₂ are prenyl groups are bisprenylated.

These monoprenylated forms and bisprenylated forms may be obtained by allowing the enzyme of the present invention to act on the compound represented by formula (III) in which both R ₁ and R ₂ are hydrogen, or may be obtained by a known synthetic method ( chemical synthesis method, enzymatic synthesis method, etc.).

The compound represented by formula (III) was newly synthesized and identified this time.

The present invention provides, in another aspect, a compound of formula (IV):

(IV)
[wherein R ₁ is hydrogen or a prenyl group and R ₂ is hydrogen or a prenyl group]
to provide a compound represented by

Compounds of formula (IV) in which R ₁ and R ₂ are both hydrogen may be used as substrates for enzymes of the invention.

A compound of formula (IV) in which either one of R ₁ and R ₂ is a prenyl group is a monoprenylated compound. Compounds of formula (IV) in which both R ₁ and R ₂ are prenyl groups are bisprenylated.

These monoprenylated forms and bisprenylated forms may be obtained by allowing the enzyme of the present invention to act on the compound represented by formula (IV) in which both R ₁ and R ₂ are hydrogen, or may be obtained by a known synthetic method ( chemical synthesis method, enzymatic synthesis method, etc.).

The compound represented by formula (IV) was newly synthesized and identified this time.

Although the present invention will be described in more detail and specifically by showing examples below, the examples are not intended to limit the scope of the present invention.

(1) General experiments ¹ H and ¹³ C NMR spectra were obtained with JEOL ECA 500 (500 MHz for ¹ H NMR), JEOL ECX 400 P (400 MHz for ¹ H NMR), JEOL ECS 400 (400 MHz for ¹ H NMR), or Buruka AVANCE Neo (500 MHz for ¹ H NMR). Chemical shifts are expressed in δ (ppm) relative to the residual solvent peak as internal standard (DMSO-d6, 1H d 2.50, 13C d 39.5). ESI-MS spectra were measured on a Thermo Scientific Exactive mass spectrometer or SHIMADZU LCMS-2020 spectrophotometer. Optical rotations were measured with a JASCO P-1030 polarimeter. High performance liquid chromatography (HPLC) was performed using a Shimadzu HPLC system equipped with an LC-20AD intelligent pump. LC-MS experiments were performed using an amaZon SL-NPC (Bruker Daltonics). Fragmentation of precursor ions was performed with an amaZon SL-NPC at an amplitude of 1.0 V using helium gas. Plasmid extraction was performed using the GenElute ^™ Plasmid Miniprep Kit (Sigma).

(2) Microcystis aeruginosa NIES-88 culture, DNA extraction, and genome sequencing Microcystis aeruginosa NIES-88 was obtained from the National Institute for Environmental Studies. Microcystis aeruginosa NIES-88 was cultured in BG-11 medium with aeration (filtered air, 0.3 L/min) at 25° C. under 250 μE/m 2 s1 illumination with 12 L:12 D cycles. After 4-5 weeks of culture, cells were harvested by continuous flow centrifugation (10000 rpm). First, macromolecular DNA was extracted by crushing frozen cells using a mortar and pestle. The cell powder was then dissolved in CTAB buffer (3% CTAB, 1.4 M NaCl, 0.2% β-mercaptoethanol, 20 mM EDTA, 100 mM Tris pH 8, RNase A) and incubated at 50°C for 30 minutes. After that, 750 μL of chloroform was added, gently inverted, and centrifuged at 15000 rpm for 5 minutes. The resulting top layer was slowly transferred to a centrifuge tube containing 200 μL of isopropanol. After the appearance of white filaments (precipitated DNA), the precipitated DNA was collected and used for genomic sequencing.

(3) Isolation of Argicyclamide A (1), B (2), and C (3) from Microcystis aeruginosa NIES-88

Argicyclamide A (1): R ₁ = R ₂ = dimethylallyl Argicyclamide B (2): R ₁ = dimethylallyl, R ₂ = H
algicyclamide C (3): R ₁ =R ₂ =H
(Argicyclamide is named by the inventors)

Lyophilized cells (10.2 g from 50 L of 4-5 week culture) were homogenized and extracted with methanol (200 mL×3). The concentrated extract was partitioned between water and ethyl acetate, and the ethyl acetate layer was further partitioned between methanol and hexanes. Both the water crude extract (533.0 mg) and the ethyl acetate crude extract (55.6 mg) were applied to ODS (YMC-Gel, 150 microns) equipped with an aqueous methanol elution system. The methanol eluted fraction was subjected to HPLC (40% MeCN with 0.1% TFA for 0-12 minutes) using C18 (Wakosil-II 5C18 AR, 20×250 mm, UV detection wavelength 215 nm, flow rate 4.0 mL/min). Purification in 57% MeCN with 0.1% TFA for 12-50 min) gave 1 (23.0 mg), 2 (2.9 mg) and 3 (2.6 mg).
Argicyclamide A (1): colorless oil; [α]D ²⁵ -53.8 (c 0.26, MeOH); HRESI(+)MS m/z 1058.7128 [M + H]+; calcd. for C ₅₇ H ₉₂ N ₁₁ O ₈ + 1058.7125.
Algicyclamide B (2): colorless oil; [α]D ²⁵ -49.9 (c 0.20, MeOH); HRESI(+)MS m/z 990.6495 [M + H]+; calcd. for C ₅₂ H ₈₄ N ₁₁ O ₈ + 990.6499.
Argicyclamide C (3): colorless oil; [α]D ²⁵ _-49.3 (c 0.19, MeOH); _HRESI (+)MS m/z _922.5873 [M + H]+; calcd. ₈ + 922.5873.

(4) Construction of plasmid Designed primers:
AgcF-F: cgcggatcccatatgttgaaaagcaacaaaaag (SEQ ID NO: 3)
AgcF-R: ccggaattcctagagcagataatatagattgagattc (SEQ ID NO: 4)
was used to amplify a DNA fragment encoding the enzyme of the present invention (referred to as AgcF) from the genomic DNA of Microcystis aeruginosa NIES-88 using KOD One (registered trademark) PCR Master Mix (TOYOBO).
A DNA fragment encoding AgcF was inserted into the multiple cloning site of pUC19 using BamHI and EcoRI to obtain AgcF-pUC19. After confirming the sequence, the AgcF insert fragment was ligated into the NdeI and EcoRI sites of pCold-II to obtain AgcF-pCold-II.

(5) Preparation of Recombinant AgcF AgcF-pCold-II was introduced into E. coli BL21 harboring the chaperone expression plasmid pGro7. 2xYT medium containing 50 μg/mL kanamycin and 30 μg/mL chloramphenicol was inoculated with a single colony and grown overnight at 37°C. 1% of the overnight culture was transferred to 200 mL of 2xYT medium containing 50 μg/mL kanamycin and 30 μg/mL chloramphenicol and 4 mg/mL L-arabinose and then incubated for 3 hours at 37°C. . AgcF gene expression was induced by adding IPTG to a final concentration of 0.1 mM and the cells were cultured overnight at 16°C. Cells were collected by centrifugation (3500×g, 10 minutes) and disrupted with an ultrasonic homogenizer. After removing debris by centrifugation (17000×g, 10 min), the fraction containing soluble protein was applied to a Ni-NTA affinity column (Merck Millipore). The column was washed with washing buffer and eluted with washing buffer containing 500 mM imidazole. The column was connected to an Amicon Ultra 0.5 mL filter (Merck Millipore). The concentration of the resulting protein solution was measured using a Bio-Rad protein assay kit. The amino acid sequence of the obtained recombinant AgcF is shown in SEQ ID NO:5.

(6) Total Synthesis of Argicyclamide C (3)

2-Chlorotriethyl resin S1 (100 mg, 0.160 mmol) in a Libra tube was swelled with CH ₂ Cl ₂ for 30 minutes, then excess solvent was removed. The resin was added to a solution of Fmoc-l-Ile-OH (17.5 mg, 0.05 mmol) and i _- Pr NEt (26 μL, 0.15 mmol) in CH ₂ Cl ₂ (0.5 mL). , and stirred at 37° C. for 1 hour. The reaction mixture was filtered, washed with DMF (x 3), CH ₂ Cl ₂ (x 3), methanol, Ac ₂ O/CH ₂ Cl ₂ (=1:4), and then vacuum dried for 1 hour. to obtain Fmoc-L-Ile-2-chlorotrityl resin S2.
S2 was swollen in CH ₂ Cl ₂ for 2 hours and subjected to 7 cycles [Fmoc-L-Phe-OH, Fmoc-L-Val-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Pro-OH , Fmoc-L-Pro-OH, Fmoc-L-Leu-OH, Fmoc-L-Val-OH] were subjected to a solid-phase peptide synthesis protocol (steps 1-4 below) to obtain resin-bound octapeptide S3. Obtained.

Solid Phase Peptide Synthesis Protocol Step 1: The Fmoc group of the solid phase supported peptide was removed by using a 20% piperidine/DMF solution (10 min, room temperature).
Step ₂ : The resin in the reaction vessel was washed with DMF (x3) and CH2Cl2 ₍ x3).
Step 3: To a solution of F-moc protected building block (4 eq) was added DIC (4 eq) in NMP and Oxyma (4 eq in DMF). After 2-3 minutes of preactivation, the mixture was poured into the reaction vessel. The resulting mixture was stirred for 30 minutes.
Step 4: The resin in the reaction vessel was washed with DMF ₍ x3) and CH2Cl2 ₍ x3). Amino acids were enriched onto the solid phase support by repeating steps 1-4.

CH ₂ Cl ₂ /(CF ₃ )CHOH (=70:30) (2.0 mL) was added to peptide S3, stirred for 20 minutes and the reaction product was filtered. This procedure was repeated twice. The filtrate was dried under vacuum to give crude peptide S4, which was used in the next step without further purification. S4 was dissolved in DMF/CH ₂ Cl ₂ (=10:90) (50 mL), and 2,6-dimethylpyridine (28 μL, 0.2 mmol), HOAt (13.5 μL, 0.2 mmol) were added to the solution. 1 mmol) and PyBOP (52.0 mg, 0.1 mmol) were added. After stirring overnight at 40° C., the reaction mixture was extracted with EtOAc (×3), washed with brine, dried over MgSO ₄ and concentrated to give crude peptide S5. TFA/phenol/anisole/DODT (=90:5:3:2) (2.0 mL) was added to the S5 residue and stirred for 30 minutes, then the reaction mixture was filtered. The filtrate was diluted with Et ₂ O (12 mL), chilled (−30° C.), and then centrifuged at 3500×g for 10 min at 4° C. to give crude peptide 3. Crude peptide 3 was purified by reversed-phase HPLC (Wakosil-II 5C18 AR, 20 x 250 mm column) (eluted with MeCN + 0.1% TFA: H ₂ O + 0.1% TFA (40:60)) to give peptide 3 (4.3 mg over 19 steps, 9.3% yield) was obtained as a white foam.
LC-MS results were compared for synthesized peptide 3, algicyclamide C isolated from Microcystis aeruginosa NIES-88 (see (3) above) (FIG. 1). Both peaks were seen at the same retention time. Also, when both were mixed, one peak was observed at the same retention time.
¹ H NMR (500 MHz, DMSO-d6) and ¹³ C NMR (125 MHz, DMSO-d6) results for synthesized peptide 3, algicyclamide C isolated from Microcystis aeruginosa NIES-88 (see (3) above). were compared (Figs. 2 and 3, respectively). In both ¹ H NMR and ¹³ C NMR, the chart patterns of synthesized peptide 3 and algicyclamide C isolated from Microcystis aeruginosa NIES-88 matched.
These results confirmed that the synthesized peptide 3 was the same substance as algicyclamide C isolated from Microcystis aeruginosa NIES-88.

(7) Conversion of Argicyclamide C (3) to Argicyclamide A (1) by the enzyme (AgcF) of the present invention (lower scheme)

Using dimethylallyl diphosphate (DMAPP) as a prenyl group donor and using algicyclamide C (the above synthetic product 3) as a prenyl group acceptor, prenyl group transfer by recombinant AgcF obtained by the above procedure (5) The reaction was confirmed in vitro. Reactions (50 μL) contained the following: 50 mM Tris-HCl (pH 8.0), 200 μM algicyclamide C, 1 mM DMAPP, 1 mM dithiothreitol, 500 mM NaCl, 50 mM MgCl ₂ . Recombinant AgcF was added to the reaction solution and the reaction was carried out at 37°C for 2 hours. The reaction solution was sampled over time and analyzed by reverse phase HPLC.

Figure 4 shows the evolution of substrates and reaction products. Consumption of substrate 3 and production of bis-prenylated product 1 were confirmed with the passage of reaction time. Formation of the mono-prenylated product 2 was also confirmed in the first half of the reaction (5 to 30 minutes). Only the substrate 3 peak was seen when the reaction was performed with AgcF that had been heat-inactivated prior to the reaction. These results confirmed that the prenyl group was indeed transferred to the arginine residue of 3 by AgcF to produce 1.

(8) Studies on Amino Acids in Prenyl Group Acceptor Peptides—Substrate Selectivity of the Enzyme of the Present Invention (AgcF) As can be seen from the above experimental results, AgcF acts twice on arginine residues in peptides. Thus, it is a novel enzyme that catalyzes bisprenylation of arginine residues. It has also been shown that AgcF does not catalyze the prenylation of lysine, serine, threonine, tryptophan, and tyrosine residues in peptides (data not shown). Therefore, more detailed substrate selectivity of AgcF was studied. In this experiment, the selectivity for sequences other than arginine in the substrate peptide was investigated.

A cyclic peptide was synthesized in which each amino acid residue contained in the sequence of algicyclamide C was replaced with alanine, and each peptide was reacted under the same conditions as in (7). The results are shown in FIG. Bisprenylation proceeded predominantly for most of the substrates, and the production of the intermediate monoprenyl form was also confirmed. However, the progress of the reaction was not observed only when the proline located next to arginine was replaced with alanine. These results indicate that prolines adjacent to arginine are important for the catalytic activity of AgcF, and that AgcF has a tolerant selectivity with respect to sequences other than prolines.

(9) Mutant of the enzyme (AgcF) of the present invention that catalyzes monoprenylation A mutation of AgcF in which the 167th histidine residue (H167) from the N-terminus of AgcF is replaced with an alanine residue using a genetic recombination method body (AgcF_H167A) was obtained. Using this mutant, a prenylation reaction of algicyclamide C was performed under the same conditions as in (7). The results are shown in FIG. On the HPLC chart of the enzyme addition reaction system, a new peak was observed at a retention time of about 6.4 minutes. Mass spectrometry (MS) revealed that this new product was monoprenylated at the arginine residue of Argicyclamide C. No peak of the bisprenylated form was observed in the HPLC chart of the enzyme addition reaction system. These results show that H167 of AgcF is an important residue for bisprenylation of arginine residues, and that substitution of H167 of AgcF with other amino acids suppresses bisprenylation and accumulates monoprenylated forms. rice field.

(10) Prenylation reaction with AgcF homolog UHCC0183PT (i) Preparation of recombinant UHCC0183PT Base sequence codon-optimized for Escherichia coli with reference to published sequence (WP_168465419.1) of AgcF homolog possessed by Aphanizomenon sp. UHCC 0183 was synthesized and inserted into the NdeI/HindIII sites of pColdII to generate the N-terminal His-tag fusion protein expression plasmid UHCC0183PT-pColdII. A recombinant UHCC0183PT protein was prepared in the same manner as in "(5) Preparation of recombinant AgcF" above. The amino acid sequence of recombinant UHCC018PT is shown in SEQ ID NO:8.
(ii) Substrate for enzyme reaction A substrate (prenyl group acceptor) shown in the following formula was synthesized and used.

Substrate synthesis was performed in the same manner as in "(6) Argicyclamide C (3) Total Synthesis" above.
(iii) Prenylation reaction with UHCC0183PT Using recombinant UHCC0183PT, the above substrate and DMAPP, The enzymatic reaction was performed under the conditions.
The reaction was analyzed by UPLC. As shown in the UPLC chromatogram of FIG. 7A, monoprenylated and bisprenylated forms were produced in an enzyme-dependent manner. The substrate was eluted at 4.7 minutes, the monoprenylated form at 5.3 minutes, and the bisprenylated form at 5.9 minutes. The results of UPLC-MS analysis in FIG. 7B confirmed that each peak in the UPLC chromatogram was derived from the substrate, monoprenylated form, and bisprenylated form.

(11) Prenylation reaction with AgcF homolog PCC9443PT (i) Preparation of recombinant PCC9443PT A nucleotide sequence with codons optimized for E. coli was synthesized with reference to the published sequence (WP_002767616) of the AgcF homolog of Microcystis aeruginosa PCC 9443. . Recombinant PC9443PT was obtained in a manner similar to the preparation of recombinant UHCC0183PT described above. The amino acid sequence of recombinant PCC9443PT is shown in SEQ ID NO:11.
(ii) Substrate for enzyme reaction A substrate (prenyl group acceptor) shown in the following formula was synthesized and used.

Substrate synthesis was performed in the same manner as in "(6) Argicyclamide C (3) Total Synthesis" above.
(iii) Prenylation reaction with PCC9443PT A reaction is performed using recombinant UHCC0183PT, the above substrate and DMAPP, and the reaction is analyzed by HPLC or UPLC to confirm the prenylation product. In addition, mass spectroscopy confirms the prenylation product.

The present invention is useful in fields such as drug discovery and production of functional peptides.

SEQ ID NO: 1 shows the amino acid sequence of AgcF (wild type).
SEQ ID NO: 2 shows the base sequence of the gene encoding AgcF (wild type).
SEQ ID NO: 3 shows the nucleotide sequence of a forward primer for amplifying a DNA fragment encoding AgcF.
SEQ ID NO: 4 shows the nucleotide sequence of a reverse primer for amplifying a DNA fragment encoding AgcF.
SEQ ID NO:5 shows the amino acid sequence of recombinant AgcF.
SEQ ID NO: 6 shows the amino acid sequence of UHCC0183PT (wild type).
SEQ ID NO: 7 shows the base sequence of the gene encoding UHCC0183PT (wild type).
SEQ ID NO:8 shows the amino acid sequence of recombinant UHCC0183PT.
SEQ ID NO: 9 shows the amino acid sequence of PCC9443PT (wild type).
SEQ ID NO: 10 shows the nucleotide sequence of the gene encoding PCC9443PT (wild type).
SEQ ID NO: 11 shows the amino acid sequence of recombinant PCC9443PT.

Claims

An enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue, or an arginine or an arginine analogue, in a peptide comprising the amino acid sequence:
(a) the amino acid sequence of SEQ ID NO: 1;
(b) an amino acid sequence having 58% or more identity to the amino acid sequence of SEQ ID NO: 1, or (c) deletion or substitution of 1 to several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 , inserted or added amino acid sequence.
A nucleic acid containing the following nucleotide sequence:
(a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1;
(b) a nucleotide sequence encoding an amino acid sequence having 58% or more identity to the amino acid sequence of SEQ ID NO: 1;
(c) a nucleotide sequence encoding an amino acid sequence in which one to several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1, or (d) the amino acid shown in SEQ ID NO: 1 A base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence encoding the sequence.
An enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue in a peptide, or an arginine or an arginine analogue encoded by the nucleic acid according to claim 2.
A vector comprising the nucleic acid according to claim 2.
A method for producing an enzyme that transfers a prenyl group to an arginine residue or an arginine analogue residue in a peptide, or an arginine or an arginine analogue, comprising the steps of:
(a) introducing the nucleic acid of claim 2 or the vector of claim 4 into a cell;
(b) culturing the cells obtained in step (a); and (c) arginine residues or arginine analogue residues, or arginine or arginine analogues in peptides from the culture obtained in step (b). Obtaining an enzyme that prenylates the body.
(i) and (ii) below:
(i) a peptide containing an arginine residue or an arginine analogue residue, or an arginine or an arginine analogue (ii) an arginine residue or an arginine residue, comprising allowing the enzyme of claim 1 or 3 to act on a prenyl group-containing compound; A method for producing a peptide in which an arginine analogue residue is prenylated, or a prenylated arginine or arginine analogue.
The method according to claim 6, wherein the peptide is a cyclic peptide.
In the peptide, a proline residue is adjacent to an arginine residue or an arginine analogue residue, and the proline residue is attached through its carboxyl group to the amino group of the arginine residue or arginine analogue residue. A method according to claim 6 or 7.
The enzyme contains an amino acid sequence in which the 167th amino acid residue from the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than histidine, or the amino acid sequence shown in SEQ ID NO: 1 The method according to any one of claims 6 to 8, wherein the amino acid residue corresponding to the 167th amino acid residue is an amino acid residue other than histidine, and the prenylation is monoprenylation. .
The method according to claim 9, wherein the amino acid other than histidine is alanine.
Arginine residue or arginine analogue residue, comprising allowing the enzyme of claim 1 or 3 to act on a peptide containing arginine residue or arginine analogue residue, or a compound library containing arginine or arginine analogue residue is a prenylated peptide, or a compound library containing prenylated arginine or arginine analogues.
The method according to claim 11, wherein the peptide is a cyclic peptide.
In the peptide, a proline residue is adjacent to an arginine residue or an arginine analogue residue, and the proline residue is attached through its carboxyl group to the amino group of the arginine residue or arginine analogue residue. 13. A method according to claim 11 or 12.
The enzyme contains an amino acid sequence in which the 167th amino acid residue from the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than histidine, or the amino acid sequence shown in SEQ ID NO: 1 The method according to any one of claims 11 to 13, wherein the amino acid residue corresponding to the 167th amino acid residue is an amino acid residue other than histidine, and the prenylation is monoprenylation. .
The method according to claim 14, wherein the amino acid other than histidine is alanine.
Formula (I):

(I)
[wherein R 1 is hydrogen or a prenyl group and R 2 is hydrogen or a prenyl group]
A compound represented by
Formula (II):

(II)
[wherein R 1 is hydrogen or a prenyl group and R 2 is hydrogen or a prenyl group]
A compound represented by
Formula (III):

(III)
[wherein R 1 is hydrogen or a prenyl group and R 2 is hydrogen or a prenyl group]
A compound represented by
Formula (IV):

(IV)
[wherein R 1 is hydrogen or a prenyl group and R 2 is hydrogen or a prenyl group]
A compound represented by