JP2002500015A - Modified deacetoxycephalosporin C synthase (DAOCS) and X-ray structure - Google Patents

Abstract

  (57) [Summary] One or more three-dimensional crystal structures of deacetoxycephalosporin C synthase (DAOCS) are described. X-ray coordinates provide accurate three-dimensional information of amino acids in the DAOCS structure. Some of these are complexed with iron and / or the substrate. Using information from such structures, enzymes of the cephalosporin biosynthetic pathway, including DAOCS, the deacetylcephalosporin C synthase DAOC / DACS, which accept non-natural substrates (eg, penicillin G, V) To increase the production of β-lactam antibiotics. Such structures can be used to predict the structure of other 2-oxoglutarate-dependent enzymes, thereby allowing the design of inhibitors and new catalysts (eg, catalysts for the production of oxidized amino acids / peptides). Specific modifications of amino acid residues are proposed and exemplified.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Penicillin and cephalosporin antibiotics are produced directly by fermentation or by modification of a fermentation inducer with a β-lactam ring. Biosynthetic pathways leading to penicillins and cephalosporins have been well studied and discussed
(JE Baldwin and CJ Schofield, The Chemistry of β-lactams (MI Page
Ed.), Chapter 1, Blackie, London 1992; Ingolia and Queener, Med. Res. Rev
., 1989, 9, 245-264; Aharonowitz, Cohen and Martin, Ann. Rev. Micobiol.,
1992, 46, 461-495; Schofield, Bycroft, Baldwin, Hadju, Roach, Current O
pinion in Structural Biology, 1997, 7, 857-864). Such a biosynthetic pathway includes the following steps (FIG. 1).

[0002] 1. Conversion of the tripeptide, L-δ-α-aminoadipoyl-L-cysteinyl-D-valine (ACV), to isopenicillin N in a step catalyzed by isopenicillin N synthase (IPNS). This step is common to the biosynthesis of both penicillin and cephalosporin.

[0003] 2. Certain organisms (eg, Penicillium chrysogenum and Aspergillus nid
ulans), isopenicillin N is converted to penicillin with another side chain (usually more hydrophobic than the side chain of isopenicillin N) by replacement of its L-δ-α-aminoadipoyl side chain. This conversion is catalyzed by the amidohydrolase / acyltransferase enzyme. Examples of penicillins produced by this biosynthetic pathway include penicillin G (having a phenylacetyl side chain) and penicillin V (having a phenoxyacetyl side chain). These hydrophobic penicillins can be produced commercially by fermentation under appropriate conditions.

[0004] 3. Other organisms such as Streptomyces clavuligerus and Cephalosporium ac
remonium), isopenicillin N is epimerized to penicillin N. This reaction is catalyzed by the epimerase enzyme.

[0005] 4. In certain organisms (eg, S. clavuligerus and C. acremonium), penicillin N is converted to DAOC. This reaction is performed by some organisms (eg, Streptomyces
clavuligerus) is catalyzed by deacetoxycephalosporin C synthase (DAOCS), while in other organisms (eg, C. acremonium) it is catalyzed by deacetoxy / deacetylcephalosporin C synthase (DAOC / DACS).

[0006] 5. In certain organisms (eg, S. clavuligerus and C. acremonium), DAOC is converted to deacetylcephalosporin C (DAC). This reaction is catalyzed by deacetylcephalosporin C synthase (DACS) in some organisms (eg, S. clavuligerus), but deacetoxy / deacetylcephalosporin in other organisms (eg, C. acremonium). It is catalyzed by C synthase (DAOC / DACS).

[0007] Further biosynthesis steps have been undertaken to give other cephalosporin derivatives, for example, in C. acremonium the DAC is converted to cephalosporin C and Streptomyces spp.
In. May be converted to cephamycin C. The genes encoding each of the enzymes that catalyze steps 1-6 above have been identified and sequenced.

[0008] The penicillins, cephalosporins and their biosynthetic intermediates obtained by fermentation are useful as antibiotics or as intermediates in antibiotic production. Penicillin having a hydrophobic side chain can be used for producing cephalosporins and intermediates for producing cephalosporins. For example, penicillins (penicillin G)
And penicillin V) can be used to produce C-3 exomethylene cepham. Such cephams are used as intermediates in the production of commercially available antibiotics (eg, Cefachlor).

The enzymes IPNS, DAOCS, DACS and DAOC / DACS are members of a broad family of oxidase and oxygenase enzymes that utilize Fe (II). The majority of this family (including DAOCS, DACS and DAOC / DACS) consists of dioxygen and "prime"
(prime) In addition to a substrate (eg, penicillin N in the case of DAOCS), a 2-oxo acid (usually 2-oxoglutarate) is utilized as a co-substrate. Since IPNS does not utilize 2-oxoglutarate, it has a substantially different mechanism of action than 2-oxoglutarate-dependent oxygenase, which gives rise to a significantly different active site.

DESCRIPTION OF THE INVENTION The present invention is based on the determination of the three-dimensional crystal structure of DAOCS and the information and development derived therefrom. X-ray coordinates provide very detailed three-dimensional information about the relationship between amino acid residues in the structure of DAOCS, as well as the mode of binding of the Fe cofactor and substrate to DAOCS. The structure of DAOCS consists of DAOCS and related enzymes (DACS and
It allows the modification of penicillin / cephalosporin biosynthetic enzymes, including DAOC / DACS, and can alter their substrate and product selectivity. DAOCS structure is 2
Since they are the first to be elucidated from the family of oxoglutarate-dependent dioxygenases, they also allow the design of new inhibitors of this family of enzymes. Previously, a partial outline of the structure of IPNS complexed with manganese and of IPNS complexed with iron and ACV has been reported (Roac
h et al., Nature, 1995, 375, 700-704; Roach et al., Nature, 1997, 387, 827). The structures defined by the X-ray coordinates of IPNS complexed with manganese and of IPNS complexed with iron, ACV and / or substrate analogs are described by Baldwin, Hajdu, Roach,
Reported in GB 9621486.1 (oxygenase enzymes and methods) by Hensgens, Clifton.

A method for producing 7-aminodeacetoxycephalosporin C (7-ADCA) has been developed. In this method, for producing cephalosporin, a recombinant in which the DAOCS gene has been introduced has been used. P. chrysogenum strain is used. In particular, when adipic acid is added to these recombinant strains, adipoyl-6-APA is produced, which is converted into adipoyl-7-ADCA by DAOCS, and the adipoyl-6-APA is converted from the adipoyl-7-ADCA. Chains are removed (EPA-A-0532341; Shibata et al., Bioorg.Med.Chem.Latts, 1996, 6,
1579-1584).

The IPNS gene sequence (hence the amino acid sequence) is DAOCS, DACS and DAOC / DACS
Related to, but quite different. Although most of the folds of these enzymes (ie, most of the elements within the three-dimensional structure) are conserved, their active site structures appear to be significantly different. Conserved structural elements include a β-barrel “jelly roll” core and a specific α-helix (
Including alpha helix-10 as defined in Roach et al., Nature, 1995, 375, 700-704). The degree of similarity is insufficient to define the exact structure of DAOCS, DACS or DAOC / DACS from the structure of IPNS. To date, no model of DAOCS, DACS or DAOC / DACS based on the structure of IPNS has been reported. No detailed studies on the substrate binding of these enzymes have been reported.
In one publication (WO 97/20053) there is a description of the products obtained by modifying certain residues in DAOCS to improve the conversion of penicillin G to phenylacetyl (G) -7-aminocephalosporanic acid. Use is claimed.

The three-dimensional structure of DAOCS is defined by X-ray coordinates (structure A) described below.

Further, the high-resolution crystal structure (structure B) of the complex of the prokaryote DAOCS from S. clavuligerus with Fe (II) and 2-oxoglutarate is also described below.

[0015] The present invention is directed, in part, to the use of the structure of DAOCS, DACS or DAOCS to modify DAOC / DACS, wherein the modified enzyme is a non-natural penicillin more efficiently than the wild-type enzyme. (E.g., penicillin G and penicillin V), for the use to modify to catalyze the conversion to cephalosporins. Further aspects of the invention include:
It relates to the use of DAOCS structures for the purpose of causing microorganisms to produce non-natural products. Such products include exomethylene cephalosporins with or without α-aminoadipoyl or hydrophobic side chains (eg, phenylacetyl or phenoxyacetyl). Thus, one aspect of the present invention is: (i) making the enzyme accept (or more efficiently accept) a non-natural penicillin substrate to produce a new or commercially valuable antimicrobial; ) For use as antimicrobial agents or as intermediates in the manufacture of antimicrobial agents or commercially valuable compounds so that the modified enzyme can produce non-natural products (eg, exomethylene cepham) or Product (for example, 3-
the use of the structure of DAOCS to modify DAOCS (or the closely related enzyme DACS or DAOC / DACS) for the purpose of optimizing the production of (β-hydroxycepham).

In another aspect, the present invention provides a modified enzyme obtained by applying the above technique. These are enzymes that have significant sequence similarity (as defined herein) to DAOCS and therefore structural similarity. That is, the structure of the modified enzyme is DA
Predictable based on OCS structure. Preferably, there will be sequence similarity / identity between a majority of the modified enzymes and a major portion of the DAOCS. Compare arrays as pairs
(pairwise comparison) and subsequent sequence comparisons using single-chain cluster analysis (Roach et al., Nature, 1995, 375, 700) show that IPNS, DAOCS, DACS and DAOC
/ DACS were shown to be grouped with a standard deviation of less than 5.0 (Barton and St.
ernberg, J. Mol. Biol. 1987, 198, 327). Standard deviation values greater than 5.0, preferably greater than 6.0, indicate that the sequence alignment is correct for all or most of the secondary structural elements of the protein (Barton, Methods in Enzymol., 1990, 183,
403). Thus, they have a significantly similar sequence and therefore a similar structure. Note that other criteria may be employed to ascertain significant sequence similarity, for example,% identity or similarity of amino acids having side chains with similar physicochemical properties (Barton and Sternberg, J. Mol. Biol. 1987, 198, 327). Thus, based on sequence comparison, the structure of one enzyme (eg, DACS or DAOC / DACS) can be predicted from another closely related enzyme (eg, DAOCS). Furthermore, although the two enzymes have a structure in which most or all of the secondary structural elements are conserved, the structural difference between the two enzymes is due to the amino acids that form the secondary structural elements. It has also been recognized that it can arise from the side chains of The effect of these differences on altering the substrate / product selectivity of the compared enzymes is predictable if the three-dimensional structure of one enzyme is known.

In another embodiment, the present invention relates to an enzyme having significant sequence similarity (as defined herein) to DAOCS, wherein the side chain binding site of penicillin N or DAOC is modified. And the following sites: Thr72, Arg74, Arg75, Glu156, Leu158,
Arg160, Arg162, Leu186, Ser187, Phe225, Phe264, Arg266, Asp301, Tyr302,
At least one amino acid residue has been changed or deleted in at least one position of Val303, Asn304, and / or at least one additional amino acid residue has been added to the region 300-. Provided is an enzyme as described above, wherein the enzyme is inserted within 311 and other residues interacting with the amino acid residue may be modified to accept a change of one of the above.

This kind of modification will allow the expansion of penicillin V or G to the corresponding cephalosporin. To do this, it is desirable to increase the kcat / Km of the mutant as compared to wild-type DAOCS. From the kinetic results, the apparent kcat values for penicillin N and penicillin G are similar,
Km is shown to be significantly higher for penicillin G. Therefore, based on these analyses, it should be possible to increase kcat / Km for penicillin G by reducing the DAOCS binding constant for penicillin G.

Since the side chain binding pocket of DAOCS is made up of residues from various parts of the peptide chain, to make a better penicillin G / V expander, two or more residues Need to be modified. Nevertheless,
Some residues are more important than others. As a result of examining the interaction between the last few C-terminal residues (Thr-308 to Ala-311) of one DAOCS molecule in the crystal structure and the active site of another molecule, The binding mode of the penicillin nucleus shown in FIG. 2 of the accompanying drawings is proposed. The carboxylate group of penam C-3 occupies a position similar to that of Ala-311 from the symmetry-related molecule, probably in the active site, with Arg-162 and
It is thought that it forms an electrostatic interaction with Arg-160. In addition, the side chain of Arg-160 can form a hydrogen bond interaction with β-lactam carbonyl.

It must be kept in mind that the specificity of a protein is generally controlled by more than one amino acid. Changing the specificity of a protein in a major way would require two or more mutational changes as suggested below, even if each of the mutations contributes. In view of this, the following residues are preferably modified for penicillin development.

A) Arg-266: This residue binds to the α-aminoadipate side chain of the natural substrate and should be changed to a more hydrophobic residue (eg, Phe, Ala, Val, Leu, Ile). is there.

B) Thr-72: This residue should be changed to a hydrophobic residue (eg, Val, Leu, Ile, Phe, Ala) to aid in binding to the hydrophobic side chain of penicillin G . This would be effective in combination with other mutations.

C) Arg-74: This residue is a neutral or hydrophobic residue (Phe, Tyr, Val, Leu, Ile, Al
a) can be changed advantageously. Since Arg-75 forms a hydrogen bonding network with Arg-74, further modifications of Arg-75 may be necessary.

D) Glu-156: This residue binds to the α-aminoadipate side chain. This is Ala, Val
, Leu, Ile, Phe, Tyr, Trp, Asn, Gln, Ser.

E) The side chains of Leu-158, Asn-301 and Tyr-302 form part of the binding pocket for penicillin side chains and can be advantageously modified to be more hydrophobic .

F) Asn-304: This residue binds to the amide that connects the side chain to the penumu nucleus. Modifications to promote penicillins with or without shortened side chains (eg, 6-
In the case of Apa, to Asp or Glu).

Note that other modifications can be used to build some or all of the side chain binding pockets by hydrogen bonding or other interactions.

In constructing a hydrophobic binding pocket for penicillin side chains, the insertion or deletion of residues into the DAOCS sequence can also be used. Insertion of hydrophobic residues into the C-terminal region (residues 300-311, especially residues 301-303) may help construct a hydrophobic binding pocket for penicillin side chains.

In another embodiment, the present invention relates to an enzyme having significant sequence similarity (as defined herein) to DAOCS, wherein the penicillin N or DAOC penicillin / cephalosporin binding The site has been modified and the following amino acid residue: Ile8
8, Arg160, Arg162, Phe164, Met180, Thr190, Ile192, Phe225, Pro241, Val24
5, at least one of Val262, Phe264, Asn304, Ile305, Arg306, Arg307 has been changed or deleted and / or at least one additional amino acid residue has been inserted into region 300-311. And the other enzyme interacting with the amino acid residue may be modified to accept one of the above.

For further discussion of this embodiment, see Nature, Vol. 394, pp. 805, published August 20, 1998.
-809, which is hereby incorporated by reference.

Another aspect of the present invention is directed to DAOCS (or a structurally related protein) to produce modified oxidized compounds, with the goal of allowing the modified enzyme to accept non-β-lactam substrates. -Oxoglutarate-dependent dioxygenase) to modify the active site of DAOCS. Oxidized amino acids (eg, 4-hydroxyproline, hydroxylysine, hydroxyaspartic acid, etc.) are useful as synthetic intermediates in producing valuable materials. By using the structure of DAOCS, specific residues can be targeted for modification, so that oxidized amino acids or peptides can be produced using modified enzymes. The method may include the following residue modifications. That is, Arg74, Glu156, L
eu158, Arg160, Arg162, Leu186, Ser187, Phe225, Phe264, Arg266, Asp301, T
yr302, Val303, Asn304, Ile88, Arg162, Phe164, Met180, Thr190, Ile192, Pr
o241, Val245, Val262, Ile305, Arg306, Arg307.

Another aspect of the invention relates to the use of the DAOCS structure for the design of a selective inhibitor of 2-oxoglutarate-dependent dioxygenase. Prolyl-4-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, has been a target of inhibition aimed at providing a therapeutic treatment for fibrotic diseases (eg, cirrhosis, arthritis). However, none of the inhibitors have been used clinically. The reason is probably that it is difficult to achieve selective inhibition of the target enzyme compared to other enzymes, including 2-oxoglutarate-dependent enzymes. The structure of DAOCS provides a template for designing inhibitors of 2-oxoglutarate-dependent dioxygenase.

The following are two high resolution crystal structures of DAOCS from S. clavuligerus:
The structure of the apoenzyme without iron (structure A) and the structure of the complex containing Fe (II) and 2-oxoglutarate (structure B) are described. These results imply a mechanism by which the enzyme-Fe (II) complex reacts with 2-oxoglutarate and dioxygen to yield reactive ferryl species, a process common to many non-heme oxygenases. are doing. Other notable 2-oxoacid-dependent ferrous enzymes are prolyl hydroxylases involved in collagen biosynthesis, and gibberellin-3β-hydroxylase, which, when mutated, affects plant length. And clavamic acid synthase involved in the biosynthesis of the β-lactamase inhibitor clavulanic acid. Among the 2-oxo acid-dependent enzymes, DAOCS is an enzyme IPN that does not use 2-oxo acid in catalysis.
It belongs to a subfamily of members showing sequence similarity to S and 1-aminocyclopropane-1-carboxylate oxidase (ethylene forming enzyme).

The iron-free form of DAOCS crystallizes as a crystallographic trimer in space group R3. The main chain of this protein folds into a conserved jellyroll core with adjacent helices.

Coordinates and structure factors are obtained from the protein data bank (entry 1rxg, Fe (II) -2-
The oxoglutarate complex has been deposited at r1rxgsf).

Structure A is a three-dimensional structure of DAOCS.

Structure B is a complex of S. clavulige with Fe (II) and 2-oxoglutarate
High resolution crystal structure of the prokaryotic DAOCS from rus.

[0038] The peptide sequence of DAOCS is listed below along with the amino acid numbers used herein.

[0039]

[Brief description of the drawings]

FIG. 1 shows the biosynthetic pathway leading to penicillins and cephalosporins.

FIG. 2 shows the active site of DAOCS showing the binding of 2-oxoglutarate to iron and the proposed binding of penicillin N. The interaction of a particular amino acid residue with the side chain is indicated by an arrow.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 4C084 1/21 9/00 5/10 C12P 35/00 9/00 C07D 501 / 10 C12P 35/00 C12N 15/00 A // C07D 501/10 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, A, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Baldwin, Jack, Edward United Kingdom Ox 15 BH Oxfordshire, Oxford, Hinksey Hill, Bloom (72) Inventor Roach, Peter, L. Ox 13 Deep Oxfordshire, Oxford, Exeter College (72) Lloyd, Matthew, De. Ox26, UK Oxfordshire, Oxfordshire, Oxford, North Wall Road 8A (72) Inventor Harros, Carl U.K.30 Oxfordshire Oxfordshire, Oxford, Purcell Road 47 (72) Invention Anderson, Swinger Es-755 91 Upsala, Staby Malmswagen 8 (72) Inventor Hardy, Janos Es-755 91 Upsala, Staby Malmswagen 8 (72) Inventor Turvis Shervan Shell Tinga, Anke, S. Sweden S-752 25 Upsala, Russags Esplanaden 8 A (72) Inventor Valgard, Karin Sweden S-752 28 Upsala, Plansgatan 6 (72) Inventor Ramazwamy, S.S. Sweden S-752 64 Up Sarah, Copens Gartan 9F term (reference) 4B024 AA01 AA07 AA11 BA80 CA04 DA02 DA05 DA11 EA04 GA11 HA01 4B050 CC03 DD01 LL05 4B064 AE43 CA01 CA19 CC24 DA01 DA12 DA13 4B065 AA01A AABAX CA46 CA47 4C075 AA10 BB02 CC02 CC19 DD02 DD13 EE02 EE05 FF01 GG01 HH01 LL01 4C084 AA17 NA14 ZA752 ZA962 ZB112 ZC802

Claims (26)

[Claims] 1. Deacetoxycephalosporin C synthase (DAOCS) having a structure specified by the X-ray coordinates of structure A or structure B described herein. 2. X-ray coordinates as described herein, complexed with a metal (eg, iron or lead), optionally in the presence of a substrate and / or a substrate analog or inhibitor. DAOCS having the structure specified by 3. The method of claim 2, wherein said substrate is penicillin N, penicillin G, 2-oxoglutarate or diatomic oxygen, and said inhibitor is selected from N-oxalyl amino acids, pyridine-carboxylate and nitrous oxide. DAOCS described. 4. Use of the three-dimensional structure of DAOCS to modify DAOCS or other related 2-oxoglutarate-dependent enzymes. 5. The use according to claim 4, wherein the related 2-oxoglutarate-dependent enzyme is an oxygenase enzyme involved in the introduction of a 7α-methoxy group into DACS, DAOC / DACS or cephamycin C. 6. A method for accepting a non-natural substrate more efficiently than a wild-type enzyme.
6. Use according to claim 5, for modifying DAOCS, DACS or DAOC / DACS. 7. Use according to claim 5, for modifying DAOCS, DACS or DAOC / DACS to convert a natural substrate into a drug or an intermediate for the manufacture of a drug. 8. The use according to claim 6, wherein the unnatural substrate is penicillin G, penicillin V, 6-aminopenicillanic acid, amoxicillin, or penicillin having a phenylglycine or p-hydroxyphenylglycine side chain. 9. Use according to claim 6, wherein the non-natural substrate is cephalosporin. 10. The use according to claim 6, wherein the unnatural substrate is an amino acid or a peptide including amino acids constituting a protein. 11. The use according to any one of claims 6 to 8, wherein penicillin G, penicillin V, other non-natural substrates or penicillin N is converted to cephalosporin or exomethylene cephalosporin. 12. An enzyme having significant sequence similarity (as defined herein) to DAOCS, wherein the penicillin N or DAOC side chain binding site has been modified, and Site: Thr72, Arg74, Arg75, Glu156, Leu158, Arg160, A
rg162, Leu186, Ser187, Phe225, Phe264, Arg266, Asp301, Tyr302, Val303, A
At least one amino acid residue has been changed or deleted in another amino acid residue in at least one site of sn304 and / or at least one additional amino acid residue is in region 300-311. The enzyme as described above, wherein the other residue interacting with the amino acid residue may be modified to accept a change of one of the above. 13. An enzyme having significant sequence similarity (as defined herein) to DAOCS, wherein the penicillin N or DAOC penicillin / cephalosporin binding site has been modified. And the following amino acid residues: Ile88, Arg160,
Arg162, Phe164, Met180, Thr190, Ile192, Phe225, Pro241, Val245, Val262,
At least one of Phe264, Ile305, Arg306, Arg307 has been changed or deleted and / or at least one additional amino acid residue has been deleted.
Such an enzyme, wherein the enzyme has been inserted into 300-311 and other residues interacting with the amino acid residue may have been modified to accept a change of one of the above. 14. The enzyme according to claim 12, which is a mutant of DAOCS or DACS or a mutant of DAOC / DACS. 15. Is both the penicillin N or DAOC side chain binding site and the penicillin / cephalosporin binding site altered, and is at least one of the residues specified in claims 12 and 13 altered? 15. The enzyme according to any one of claims 12 to 14, wherein the enzyme is deleted. 16. The method of claim 12, wherein two or more complementary mutations have been introduced to create or delete a binding interaction, including a hydrogen bond, electrostatic or hydrophobic interaction. An enzyme according to any one of the preceding claims. A gene encoding the enzyme according to any one of claims 12 to 16. 18. A microorganism capable of expressing the gene according to claim 17 under fermentation conditions. 19. Use of the microorganism according to claim 18 for producing a β-lactam of the penicillin or cephalosporin (including cepham) family. 20. The penicillin / cephalosporin biosynthesis pathway, wherein the microorganism comprises isopenicillin N synthase, amidohydrolase / acetyltransferase, or L-δ- (aminoadipoyl) -L-cysteine-D-valine (ACV) synthetase. 20. Use according to claim 19, comprising another modified enzyme. 21. The three-dimensional structure of DAOCS is used to determine or predict the structure of other related 2-oxoglutarate-dependent enzymes or related enzymes not derived from the penicillin / cephalosporin biosynthetic pathway. A method comprising using the structural information thus obtained to modify another enzyme or to design an inhibitor of another enzyme. 22. The other related 2-oxoglutarate-dependent enzyme or related enzyme is 1-aminocyclopropane-1-carboxylate oxidase, gibberellin C-20 oxidase, flavone synthase, flavanone-3β-hydroxylase. , Hyoscyamine-6β-hydroxylase, prolyl-4-hydroxylase, prolyl-3-hydroxylase, aspartyl hydroxylase, lysylhydroxylase, proline hydroxylase, γ-butyrobetaine hydroxylase, involved in herbicide resistance mechanisms Enzymes, clavamic acid synthase, oxygenase enzymes involved in carbapenem biosynthesis, so-called ethylene-forming enzymes from Pseudomonas syringe, p-hydroxyphenylpyruvate dioxygenase, and phytosomes in human liver peroxisomes. An oxygenase enzyme involved in the oxidation of method of claim 21, wherein. 23. The method according to claim 1, wherein the other enzyme is deleted, added or substituted.
23. The method according to claim 21 or 22, wherein the modification is at one or more of the sites defined in 2 or 13 or using the following information for the design of the inhibitor: Asp185, His183 and His243 against iron. Acts as ligand; Arg258, Ser2
60 and Fe bind to 2-oxoglutarate; Met180, Phe225, Leu31 and V
al245 approaches the iron binding site; Tyr33, Arg160, Arg162, Phe164, Ile192, Gln
194, Leu204, Leu223 and Leu215 are important for building part of the active site that binds 2-oxoglutarate; Arg160 and Arg162 are important for binding substrates derived from amino acids or peptides . 24. The other enzyme is prolyl-4-hydroxylase, prolyl-hydroxylase.
24. Any one of claims 21 to 23, which is 3-hydroxylase, aspartyl hydroxylase, or lysyl hydroxylase, wherein the inhibitor is used for treating human diseases including fibrotic diseases such as cirrhosis and arthritis. the method of. 25. The method of claim 2, wherein the other enzyme is p-hydroxyphenylpyruvate dioxygenase, and wherein the inhibitor is used to treat a particular genetic disease.
24. The method according to any one of 1 to 23. 26. The method of any one of claims 21 to 23, wherein the other enzyme is involved in herbicide resistance and the information is used in designing a new herbicide to overcome the resistance problem. the method of.