CN1284997A - Modified deacetoxycephalosporin C synthase (DAOCS) and x-ray structure - Google Patents

Abstract

This paper describes the three-dimensional crystal structure of deacetoxycephalosporin C synthase (DAOCS). X-ray coordinates provide precise three-dimensional information of the amino acids in the DAOCS structure. Some of these amino acids form complexes with iron and/or substrates. Information from the structure was used to modify enzymes of the cephalosporin biosynthetic pathway including DAOCS, deacetylcephalosporin C synthase DAOC/DACS, by modifying them to accept unnatural substrates (e.g., penicillin G, V), thereby facilitating Production of β-lactam antibiotics. These structures can be used to predict the structures of other 2-oxoglutarate-dependent enzymes, thereby enabling the design of inhibitors, as well as new catalysts for the production of eg oxidized amino acids/peptides. Specific modifications to amino acid residues are suggested and exemplified herein.

Description

DeacetoxycephalosporCn Cn synthase (DAOCS) and the x-ray structure modified

Penicillin and cephalosporin antibiotics are directly by fermentation or contain the material preparation that comes from fermentation of beta-lactam ring by modification.Penicillin and cephalo bacterium biosynthetic pathway are widely studied and summarize (J.E.Baldwin and C.J.Schofield, " beta-lactam chemistry " (M.I.Page volume), the 1st chapter, Blackie, London 1992; Ingolia and Queener, medical research comment, 1989,9,245-264; Aharonowitz, Cohen and Martin, microorganism academic year is stated, and 1992,46,461-495; Schofield, Bycroft, Baldwin, Hadju, Roach, contemporary structure biology comment, 1997,7,857-864), may further comprise the steps (Fig. 1):

1. the conversion of tripeptides: in by the catalytic step of isopenicillin N synthase (IPNS), change L-δ-alpha-amino group adipyl-L-cysteinyl-D-Xie Ansuan (ACV) into isopenicillin N.This step is common in penicillin and the cephamycin biosynthesizing.

2. in some biology (routine Penicllium chrysogenum (Penicillium Chrysogenum) and Aspergillus nidulans (Aspergillus nidulans)); isopenicillin N is by exchanging its L-δ-alpha-amino group adipyl side chain and other side chain of penicillin and transformed, and wherein other side chain is stronger than isopenicillin side chain hydrophobicity usually.This transforms by hydroamidase/acyl group transfer catalysis.The example of the penicillin of biosynthetic process preparation comprises penicillin G (phenylacetyl side chain of tool) and penicillin v (a benzene oxygen of tool ethanoyl side chain) thus.These hydrophobic penicillin can be under proper condition by the fermentation commercial production.

3. (for example be with spillikin streptomycete (Streptomycesclavuligerus) and branch top spore (Cephalosporium acremonium)) in other biology, isopenicillin N is formed penicillin N by epimerization.This reaction is by the epimerization enzyme catalysis.

4. some biology (for example being with spillikin streptomycete and branch top spore), penicillin N changes DAOC into.This is reflected in some biology (example band spillikin streptomycete) by desacetoxy cephalosporin synthase (DAOCS) catalysis, in other biology (for example branch top spore) by desacetoxy/deacetyl cephalosporin C synthase (DAOC/DACS) catalysis.

5. DAOC is changed into deacetyl cephalosporin C (DAC) (for example to be with spillikin streptomycete and branch top spore) in some biology.In some biology (for example being with the spillikin streptomycete), this reacts by deacetyl cephalosporin C synthase (DACS) catalysis, in other biology (for example branch top spore), by desacetoxy/deacetyl cephalosporin C synthase (DAOC/DACS) catalysis.

Other biosynthesizing step that produces other cephalosporins derivatives also may produce, and for example, in the spore of branch top, DAC can change cephalosporin into, and in streptomycete, DAC also can change cephamycin C into.The encoding gene of every kind of enzyme in above 1-6 step of catalysis is identified and is checked order.

The penicillin of fermentation, cynnematin and their biosynthesizing intermediate can be used as the intermediate in microbiotic or the preparation microbiotic.Penicillin with hydrophobic side chain can be used for preparing the intermediate in cynnematin or the preparation cynnematin, for example penicillin (comprising penicillin G and penicillin v) can be used for preparing C-3 exomethylenecepham alkene class, this C-3 exomethylenecepham alkene for example can be used as intermediate in the cefaclor (Cefacholr) at the commercial microbiotic of preparation.

Enzyme IPNS, DAOCS, DASC and DAOC/DACS all belong to the member who utilizes oxydase and oxygenase Fe (II) to enlarge family.Major part in this family (comprising DAOCS, DACS and DAOC/DACS) utilizes 2-oxidizing acid (being generally 2-oxoglutaric acid) as the cosubstrate except that dioxygen and main substrate (for example at the penicillin N under the DAOCS situation).Because IPNS does not utilize 2-oxoglutaric acid, it has the different in essence mechanism of oxygenase that relies on 2-oxoglutaric acid, and this produces significantly different avtive spot.

The present invention

The present invention is based on information and the development determining and come from this to the three-dimensional crystalline structure of DAOCS.The X ray coordinate provide about the combination of the Fe-cofactor that concerns between the amino-acid residue in the DAOCS structure and substrate and DAOCS detailed three to information.This structure allows the relevant enzyme of DAOCS and penicillin and cynnematin biosynthesizing (comprising DACS and DAOC/DACS) is modified, with substrate and the selectivity of product that changes them.Because the structure of first discovery of dioxygenase family that the DAOCS structure is a 2-oxoglutaric acid to be relied on, they also can be used for the design of the novel inhibitors of this enzyme family.Once reported in the past that IPNS combines with manganese and IPNS and iron compound, and the part of the structure of ACV summary (Roach etc., nature, 1995,375,700-704; Roach etc., nature, 1997,387,827).Shown in the X ray coordinate, IPNS and manganese compound and and iron, the structure of the mixture that ACV and/or substrate analogue form is once at Baldwin, Hajdu, Roach, Hensgens, Clifton, report among the GB9621486,1-(oxygenase and method).

Developed the method for preparing the amino desacetoxy cephalosporin of 7-(7-ADCA), wherein the reorganization Penicllium chrysogenum bacterial strain of DAOCS gene importing is used to the production of cynnematin.If when especially hexanodioic acid being added these recombinant bacterial strains, produce adipyl-6-APA, it is converted into adipyl-7-ADCA by DAOCS, and its adipyl side chain can be removed (EPA-A-0532341, Shibata etc., biomedical chemical communication.1996，6，1579-1584)。

IPNS gene order (and aminoacid sequence) and DAOCS, DACS, the sequence of DAOC/DACS is relevant but significantly different.Folding whole elements (being the active component in the three-dimensional structure) of possible these enzymes are guarded, and the avtive spot structure is extremely significantly different.Conservative structural element may comprise β bucket gel roll (' jelly roll ') nuclear and some α spiral (comprise α spiral 10, as at Roach etc., nature 1995,375, describes in detail among the 700-704).Similar degree is not enough to determine to be derived from the DAOCS of IPNS structure, DACS, or the fine structure of DAOC/DACS.The DAOCS of report based on the IPNS structure do not arranged so far, DACS, or the model of DAOCS/DACS.A report (WO 97/20053) requires product that the modification to specific residue among the DAOCS obtains to change application in phenylacetyl (G)-7-aminocephalosporinic acid at improved penicillin G.

The three-dimensional structure of DAOCS is determined (structure A) by X ray coordinate as follows.

The high definition crystalline structure (structure B) that below also shows the mixture of the protokaryon DAOCS carry the spillikin streptomycete and Fe (II) and 2-oxoglutaric acid.

In one aspect, the present invention relates to use the DAOCS structure, thereby DAOCS or DACS or DAOC/DACS are modified the enzyme catalysis non-natural penicillin (for example penicillin G and penicillin v) when being converted into cynnematin that makes modified, more effective than wild-type enzyme.Another aspect of the present invention relates to the DAOCS structure prepares the non-natural product in microorganism application.These products comprise different methene cynnematin, and they contain or do not contain alpha-amino group adipyl or hydrophobic side chain (routine phenylacetyl or benzene oxygen acetyl).Therefore, one aspect of the present invention relates to the structural modification DAOCS (or enzyme DACS or DAOC/DACS of being closely related) that uses DAOCS, is intended to:

(ⅰ) make enzyme accept (or more effective acceptance) non-natural penicillin substrate to be used for the preparation of antimicrobial substance new or the tool commercial value.

(ⅱ) make the enzyme of modification can produce non-natural product (for example different methene cephamycin) or optimize the preparation of low amounts of product (routine 3-beta-hydroxy cephamycin), with as antibiotic or commercial with antibiotic or intermediate in the compound.

The invention provides on the other hand and use the adorned enzyme that above-mentioned technology obtains.These be contain important (the following detailed description in detail) sequence and with the enzyme of DAOCS structural similarity.Therefore the structure of these enzymes can be predicted on the DAOCS architecture basics.Preferably between the major portion of enzyme that great majority are modified and DAOCS, there is sequence similarity/identity.Sequence in the past is (Roach etc., nature, 1995,375,700) relatively, adopt the sequence paired comparison, carry out single linkage group analysis then, show IPNS, DAOCS, DACS and DAOC/DACS group have standard deviation greater than 5.0 (Barton and Sternberg, the molecular biosciences magazine, 1987,198,327).Numerical value surpasses 5.0 and preferably surpass 6.0 and mean sequence order correct (Barton, Enzymology method, 1990,183,403) in all or the most albumen secondary structure element; Therefore they have a large amount of similar sequences and so and next structure.Note also having other standard to can be used for determining the similarity of important sequence, for example have amino acid whose identity or similarity per-cent (Barton and the Sternberg of the side chain of resemblance physical chemistry characteristic.The molecular biology magazine, 1987,198,327).Therefore, on sequence basis relatively, can predict the structure of a kind of enzyme (routine DACS or DAOC/DACS) from other enzyme that is closely related (routine DAOCS).Further also recognize: although two kinds of enzymes may have wherein most of or the structure of conservative secondary structure element fully, the difference on two kinds of enzymatic structures also may be caused by the amino acid whose side chain that forms the secondary structure element.The influence that these differences of the substrate/selectivity of product of change enzyme relatively cause is predictable, in case know a kind of three-dimensional structure of enzyme.

The invention provides on the other hand with DAOCS and have the significantly enzyme of (as defined herein) sequence similarity, wherein the side chain binding site of penicillin N or DAOC is modified, and in following site, at least one amino-acid residue is changed into another amino-acid residue or disappearance: Thr72, Arg74, Arg75, Glu156, Leu158, Arg160, Arg162, Leu186, Ser187, Phe225, Phe264, Arg266, Asp301, Tyr302, Val303, Asn304; And/or at least one additional amino acid residue inserts between regional 300-311; Prerequisite is may be changed with interactional other residue of above residue, to cooperate one of above-mentioned change.

Such modification allows penicillin v or penicillin G to expand to corresponding cynnematin.For realizing this target, compare the Kcat/Km value that needs to increase mutant with wild-type DAOCS, the apparent Kcat value of kinetic results explanation penicillin N and penicillin G is similar, but the Km value of penicillin G is much higher.Therefore, analyze based on these, the binding constant of DAOCS and penicillin G reduces should make the Kcat/Km ratio increase of penicillin G become possibility.

The side chain binding pocket of DAOCS is formed by the residue of peptide chain different piece, and therefore not only a residue has to change to form better penicillin G/V expansion.Even so, some residue is more important than other residue.To in last several C-terminal residues (Thr308 to Ala-311) of DAOCS molecule and the crystalline structure between other avtive spot interactional detection shown that with the combination of penicillin nuclear, it is as shown in Figure 2 in the accompanying drawing.Blue or green nucleic C-3 carboxyl occupies and the similar position of Ala-311 from the symmetrical associated molecule of avtive spot probably, forms electrostatic interaction with Arg-162 and Arg-60.The side chain of Arg-160 and beta-lactam carbonyl form interaction of hydrogen bond.

Need remember that protein-specific is generally by surpassing an amino acid control.May need not only sudden change of following suggestion to change generally in order to change protein-specific, although every kind of sudden change is all worked.Know that therefrom the residue of expansion that preferably is used to modify penicillin is as follows:

A) this residue of Arg-266. combines with the α-An Jijiersuan side chain of natural substrate, should change the residue that has more hydrophobic property into, routine Phe, Ala, Val, Leu, Ile.

B) Thr-72. it should change hydrophobic residue into, Val for example, Leu, Ile, Phe, Ala is to help the hydrophobic side chain combination of penicillin G.Combine then more effective with other sudden change.

C) Arg-74 can usefully change into neutrality or hydrophobic residue (Phe, Tyr, Val, Leu, Ile, Ala).May be additionally need be to the modification of Arg-75, because of itself and Arg74 form hydrogen bond network.

D) this residue of Glu-156. combines with the α-An Jijiersuan side chain.It should be become Ala, Val, Leu, Ile, Phe, Tyr, Trp, Asn, Gln, among the Ser one.

E) Leu-158, the side chain of Asn-301 and Tyr-302 forms the part of penicillin side chain binding pocket, and can have more hydrophobic property through effective modification.

F) this residue of Asn-304. makes acid amides connection side chain combine with blue or green nucleic nuclear.Modify with expansion tool penicillin brachymemma or that do not have side chain (for example be changed to Asp or Glu, form 6-Apa).

Notice that other variation can be used for by hydrogen bond or other part or all of side chain binding pocket of structure that interacts.

The residue of DAOCS sequence inserts or disappearance also can be used for making up hydrophobic binding pocket with the penicillin side chain.Hydrophobic residue is inserted into C-terminal zone (residue 300-311, particularly 301-303), can help the structure of the hydrophobic binding pocket of penicillin side chain.

The invention provides on the other hand with DAOCS and have the significantly enzyme of (as defined herein) sequence similarity, wherein the penicillin of penicillin N or DAOC/cynnematin binding site is through modifying, and at least a following amino-acid residue is changed or lacks: Ile88, Arg160, Arg162, Phe164, Met180, Thr190, Ile192, Phe225, Pro241, Val245, Val262, Phe264, Asn304, Ile305, Arg306, Arg307; And/or at least one additional amino acid residue is inserted into regional 300-311; Prerequisite is may be changed to cooperate above change with interactional other residue of above residue.

Nature the 394th volume is seen in the further discussion of this respect, the 805-809 page or leaf, and 1998,8,20 publish, in this income as a reference.

The present invention relates to the structure of using DAOCS on the other hand, changing its avtive spot (or avtive spot of the dioxygenase that relies on of the 2-oxoglutaric acid of structurally associated), thereby makes the enzyme of modification accept non-beta-lactam substrate, produces valuable oxygenated compound.The amino acid of oxidation (4-Hydroxyproline for example, hydroxylysine, hydroxyl aspartic acid and other) can act on synthetic intermediate in the production of valuable substance.The structure of utilization DAOCS, but target is decided the specificity residue to be used for modification, so that the enzyme after modifying can be used for the amino acid or the peptide of production oxidation.This process can comprise the modification to following residue.

Arg74，Glu156，Leu158，Arg160，Arg162，Leu186，Ser187，Phe225，Phe264，Arg266，Asp301，Tyr302，Val303，Asn304，Ile88，Arg162，Phe164，Met180，Thr190，Ile192，Pro241，Val245，Val262，Ile305，Arg306，Arg307。

The present invention relates to the application of DAOCS structure in the design of the selective depressant of the dioxygenase that 2-oxoglutaric acid relies on the other hand.For the treatment to fibrotic disease (for example liver cirrhosis, sacroiliitis) is provided, the dioxygenase prolyl 4-hydroxylase that 2-oxoglutaric acid relies on is the target that suppresses.But the inhibitor that does not still have clinical use is likely owing to be difficult to realize be different from the selectivity inhibition to the target enzyme of other enzyme (comprising the enzyme that 2-oxoglutaric acid relies on).The structure of DAOCS provides template for the inhibitor of the dioxygenase that the design 2-oxoglutaric acid relies on.

Below provide 2 kinds of high definition crystalline structure of the DAOCS that carries the spillikin streptomycete: structure of iron-free apoenzyme (structure A) and the complex structure (structure B) that forms with Fe (II) and 2-oxoglutaric acid.The result has shown a kind of mechanism, and to produce active iron, this is the total processes of many non-heme oxygenases by this mechanism enzyme-Fe (II) complex body and 2-oxoglutaric acid and dioxygen reaction.The ferrous enzyme that other important 2-oxygen (generation) acid relies on is for participating in the biosynthetic prolyl hydroxylase of collagenase; Plant hormones regulators,gibberellins 3 B-hydroxylases; its sudden change can influence the length of axis; with excellent alkanoic acid (Clavaminic acid) synthase, this enzyme participates in the biosynthesizing of beta-lactamase inhibitor rod alkanoic acid.In 2-oxoacid dependent enzyme family, DAOCS belongs to a subtribe, and its member's demonstration and IPNS and 1-amino-cyclopropane-1-carboxylate oxydase (ethene formation enzyme) has sequence similarity, and these enzymes do not use the 2-oxoacid in catalytic process.

The iron-free form of DAOCS is in the space radicals R ₃Under the situation with the crystallization of crystal trimeric form.The albumen backbone fold is the gel rolling nuclear of conservative tool side spiral.

Coordinate and structure factor are stored in albumen database (for Fe (II)-2-oxoglutaric acid complex body, being registered as 1rxg and rlrxgsf).

Description of drawings

The biosynthetic pathway of Fig. 1 penicillin and cephamycin.

Fig. 2 is the active sites point diagram of DAOCS, shows combining and the penicillin N combination of suggestion of 2-oxoglutaric acid and iron.Arrow marks the interaction of particular amino acid residue side chain.

Structure A is the three-dimensional structure of DAOCS.

Structure B is the high definition crystalline structure of conduct with the protokaryon DAOCS that carries the spillikin streptomycete of the composite form of Fe (II) and 2-oxoglutaric acid.

the peptide sequence of DAOCS, (using numbering used herein) is as follows: Met, Asp, Thr, Thr, Val, Pro, Thr, Phe, Ser, Leu, 10Ala, Glu, Leu, Gln, Gln, Gly, Leu, His, Gln, Asp, 20Glu, Phe, Arg, Arg, Cys, Leu, Arg, Asp, Lys, Gly, 30Leu, Phe, Tyr, Leu, Thr, Asp, Cys, Gly, Leu, Thr, 40Asp, Thr, Glu, Leu, Lys, Ser, Ala, Lys, Asp, Leu, 50Val, Ile, Asp, Phe, Phe, Glu, His, Gly, Ser, Glu, 60Ala, Glu, Lys, Arg, Ala, Val, Thr, Ser, Pro, Val, 70Pro, Thr, Met, Arg, Arg, Gly, Phe, Thr, Gly, Leu, 80Glu, Ser, Glu, Ser, Thr, Ala, Gln, Ile, Thr, Asn, 90Thr, Gly, Ser, Tyr, Ser, Asp, Tyr, Ser, Met, Cys, 100Tyr, Ser, Met, Gly, Thr, Ala, Asp, Asn, Leu, Phe, 110Pro, Ser, Gly, Asp, Phe, Gly, Arg, Ile, Trp, Thr, 120Gln, Tyr, Phe, Asp, Arg, Gln, Tyr, Thr, Ala, Ser, 130Arg, Ala, Val, Ala, Arg, Glu, Val, Leu, Arg, Ala, 140Thr, Gly, Thr, Glu, Pro, Asp, Gly, Gly, Val, Glu, 150Ala, Phe, Leu, Asp, Cys, Glu, Pro, Leu, Leu, Arg, 160Phe, Arg, Tyr, Phe, Pro, Gln, Val, Pro, Glu, His, 170Arg, Ser, Ala, Glu, Glu, Gln, Pro, Leu, Arg, Met, 180Ala, Pro, His, Tyr, Asp, Leu, Ser, Met, Val, Thr, 190Leu, Ile, Gln, Gln, Thr, Pro, Cys, Ala, Asn, Gly, 200Phe, Val, Ser, Leu, Gln, Ala, Glu, Val, Gly, Gly, 210Ala, Phe, Thr, Asp, Leu, Pro, Tyr, Arg, Pro, Asp, 220Ala, Val, Leu, Val, Phe, Cys, Gly, Ala, Ile, Ala, 230Thr, Leu, Val, Thr, Gly, Gly, Gln, Val, Lys, Ala, 240Pro, Arg, His, His, Val, Ala, Ala, Pro, Arg, Arg, 250Asp, Gln, Ile, Ala, Gly, Ser, Ser, Arg, Thr, Ser, 260Ser, Val, Phe, Phe, Leu, Arg, Pro, Asn, Ala, Asp, 270Phe, Thr, Phe, Ser, Val, Pro, Leu, Ala, Arg, Glu, 280Cys, Gly, Phe, Asp, Val, Ser, Leu, Asp, Gly, Glu, 290Thr, Ala, Thr, Phe, Gln, Asp, Trp, Ile, Gly, Gly, 300Asn, Tyr, Val, Asn, Ile, Arg, Arg, Thr, Ser, Lys, 310Ala, 311

Sequence Table <110>, Degussa - stock company in Wales <120> eno gene encoding nucleotide sequence of new <130> 990152 BT <140> <141> <160> 2 <170> Patent In Ver. 2.1 <210> 1 <211> 1578 <212> DNA <213> Corynebacterium glutamicum <220> <221> CDS <222> (151). . (1425) <400> 1 ggctggggat atgggtagtt ttcgccacta atttcaactg attgcctcat cgaaacaaga 60 ttcgtgcaac aattgggtgt agacgtgatt gaagacattt gatcacgtga ataattctag 120 ttagctccca agttggcata ggaggccaca gtg gct gaa atc ​​atg cac gta ttc 174 Val Ala Glu Ile Met His Val Phe 15 gct cgc gaa att ctc gac tcc cgc ggt aac cca acc gtc gag gca gag 222 Ala Arg Glu Ile Leu Asp Ser Arg Gly Asn Pro Thr Val Glu Ala Glu 101520 gtt ttc ctg gat gac ggt tcc cac ggt gtc gca ggt gtt cca tcc ggc 270 Val Phe Leu Asp Asp Gly Ser His Gly Val Ala Gly Val Pro Ser Gly 25303540 gca tcc acc ggc gtc cac gag gct cat gag ctg cgt gac ggt ggc gat 318 Ala Ser Thr Gly Val His Glu Ala His Glu Leu Arg Asp Gly Gly Asp 45 50 55 5 cgc tac ctg ggc aag ggc gtt ttg aag gca gtt gaa aac gtc aac gaa 366 Arg Tyr Leu Gly Lys Gly Val Leu Lys Ala Val Glu Asn Val Asn Glu 60 65 70 gaa atc ​​ggc gac gag ctc gct ggc cta gag gct gac gat cag cgc ctc 414 Glu Ile Gly Asp Glu Leu Ala Gly Leu Glu Ala Asp Asp Gln Arg Leu 75 80 85 atc gac gaa gca atg atc aag ctt gat ggc acc gcc aac aag tcc cgc 462 Ile Asp Glu Ala Met Ile Lys Leu Asp Gly Thr Ala Asn Lys Ser Arg 9095100 ctg ggt gca aac gca atc ​​ctt ggt gtt tcc atg gct gtt gca aag gct 510 Leu Gly Ala Asn Ala Ile Leu Gly Val Ser Met Ala Val Ala Lys Ala 105 110 115 120 gct gct gat tcc gca ggc ctc cca ctg ttc cgc tac atc ggt gga cca 558 Ala Ala Asp Ser Ala Gly Leu Pro Leu Phe Arg Tyr Ile Gly Gly Pro 125130135 aac gca cac gtt ctt cca gtt cca atg atg aac atc atc aac ggt ggc 606 Asn Ala His Val Leu Pro Val Pro Met Met Asn Ile Ile Asn Gly Gly 140 145 150 gct cac gct gac tcc ggt gtt gac gtt cag gaa ttc atg atc gct cca 654 Ala His Ala Asp Ser Gly Val Asp Val Gln Glu Phe Met Ile Ala Pro 155160165 atc ggt gca gag acc ttc tct gag gct ctc cgc aac ggc gcg gag gtc 702 Ile Gly Ala Glu Thr Phe Ser Glu Ala Leu Arg Asn Gly Ala Glu Val 170175180 tac cac gca ctg aag tcc gtc atc aag gaa aag ggc ctg tcc acc gga 750 Tyr His Ala Leu Lys Ser Val Ile Lys Glu Lys Gly Leu Ser Thr Gly 185190195200 ctt ggc gat gag ggc ggc ttc gct cct tcc gtc ggc tcc acc cgt gag 798 Leu Gly Asp Glu Gly Gly Phe Ala Pro Ser Val Gly Ser Thr Arg Glu 205210215 gct ctt gac ctt atc gtt gag gca atc ​​gag aag gct ggc ttc acc cca 846 Ala Leu Asp Leu Ile Val Glu Ala Ile Glu Lys Ala Gly Phe Thr Pro 220225230 ggc aag gac atc gct ctt gct ctg gac gtt gct tcc tct gag ttc ttc 894 Gly Lys Asp Ile Ala Leu Ala Leu Asp Val Ala Ser Ser Glu Phe Phe 235240245 aag gac ggc acc tac cac ttc gaa ggt ggc cag cac tcc gca gct gag 942 Lys Asp Gly Thr Tyr His Phe Glu Gly Gly Gln His Ser Ala Ala Glu 250 255 260 atg gca aac gtt tac gct gag ctc gtt gac gcg tac cca atc ​​gtc tcc 990 Met Ala Asn Val Tyr Ala Glu Leu Val Asp Ala Tyr Pro Ile Val Ser 265270275280 atc gag gac cca ctg cag gaa gat gac tgg gag ggt tac acc aac ctc 1038 Ile Glu Asp Pro Leu Gln Glu Asp Asp Trp Glu Gly Tyr Thr Asn Leu 285290295 acc gca acc atc ggc gac aag gtt cag atc gtt ggc gac gac ttc ttc 1086 Thr Ala Thr Ile Gly Asp Lys Val Gln Ile Val Gly Asp Asp Phe Phe 300305310 gtc acc aac cct gag cgc ctg aag gag ggc atc gct aag aag gct gcc 1134 Val Thr Asn Pro Glu Arg Leu Lys Glu Gly Ile Ala Lys Lys Ala Ala 315320325 aac tcc atc ctg gtt aag gtg aac cag atc ggt acc ctc acc gag acc 1182 Asn Ser Ile Leu Val Lys Val Asn Gln Ile Gly Thr Leu Thr Glu Thr 330335340 ttc gac gct gtc gac atg gct cac cgc gca ggc tac acc tcc atg atg 1230 Phe Asp Ala Val Asp Met Ala His Arg Ala Gly Tyr Thr Ser Met Met 345350355360 tcc cac cgt tcc ggt gag acc gag gac acc acc att gct gac ctc gca 1278 Ser His Arg Ser Gly Glu Thr Glu Asp Thr Thr Ile Ala Asp Leu Ala 365370375 gtt gca ctc aac tgt ggc cag atc aag act ggt gct cca gca cgt tcc 1326 Val Ala Leu Asn Cys Gly Gln Ile Lys Thr Gly Ala Pro Ala Arg Ser 380385390 gac cgt gtc gca aag tac aac cag ctt ctc cgc atc gag cag ctg ctt 1374 Asp Arg Val Ala Lys Tyr Asn Gln Leu Leu Arg Ile Glu Gln Leu Leu 395400405 ggc gac gcc ggc gtc tac gca ggt cgc agc gca ttc cca cgc ttt cag 1422 Gly Asp Ala Gly Val Tyr Ala GIy Arg Ser Ala Phe Pro Arg Phe Gln 410415420 ggc taaataaaag cgcttttcga cgcccggtaa cctcaaggtt gccgggcgtc 1475 Gly 425 gttgccttac tactgttact ggtgtgacta tgatcgagga ttatggcaaa gcagaagaaa 1535 actcataaag gccttgttcc tgtctcaagc agggaacgtg ctt 1578 <210> 2 <211> 425 <212> PRT <213> Corynebacterium glutamicum <400> 2 Val Ala Glu Ile Met His Val Phe Ala Arg Glu Ile Leu Asp Ser Arg 151015 Gly Asn Pro Thr Val Glu Ala Glu Val Phe Leu Asp Asp Gly Ser His 202530 Gly Val Ala Gly Val Pro Ser Gly Ala Ser Thr Gly Val His Glu Ala 35 40 45 His Glu Leu Arg Asp Gly Gly Asp Arg Tyr Leu Gly Lys Gly Val Leu 50 55 60 Lys Ala Val Glu Asn Val Asn Glu Glu Ile Gly Asp Glu Leu Ala Gly 65 70 75 80 Leu Glu Ala Asp Asp Gln Arg Leu Ile Asp Glu Ala Met Ile Lys Leu 85 90 95 Asp Gly Thr Ala Asn Lys Ser Arg Leu Gly Ala Asn Ala Ile Leu Gly 100 105 110 Val Ser Met Ala Val Ala Lys Ala Ala Ala Asp Ser Ala Gly Leu Pro 115120125 Leu Phe Arg Tyr Ile Gly Gly Pro Asn Ala His Val Leu Pro Val Pro 130135140 Met Met Asn Ile Ile Asn Gly Gly Ala His Ala Asp Ser Gly Val Asp 145150155160 Val Gln Glu Phe Met Ile Ala Pro Ile Gly Ala Glu Thr Phe Ser Glu 165170175 Ala Leu Arg Asn Gly Ala Glu Val Tyr His Ala Leu Lys Ser Val Ile 180185190 Lys Glu Lys Gly Leu Ser Thr Gly Leu Gly Asp Glu Gly Gly Phe Ala 195200205 Pro Ser Val Gly Ser Thr Arg Glu Ala Leu Asp Leu Ile Val Glu Ala 210215220 Ile Glu Lys Ala Gly Phe Thr Pro Gly Lys Asp Ile Ala Leu Ala Leu 225230235240 Asp Val Ala Ser Ser Glu Phe Phe Lys Asp Gly Thr Tyr His Phe Glu 245250255 Gly Gly Gln His Ser Ala Ala Glu Met Ala Asn Val Tyr Ala Glu Leu 260265270 Val Asp Ala Tyr Pro Ile Val Ser Ile Glu Asp Pro Leu Gln Glu Asp 275280285 Asp Trp Glu Gly Tyr Thr Asn Leu Thr Ala Thr Ile Gly Asp Lys Val 290295300 Gln Ile Val Gly Asp Asp Phe Phe Val Thr Asn Pro Glu Arg Leu Lys 305310315320 Glu Gly Ile Ala Lys Lys Ala Ala Asn Ser Ile Leu Val Lys Val Asn 325330335 Gln Ile Gly Thr Leu Thr Glu Thr Phe Asp Ala Val Asp Met Ala His 340345350 Arg Ala Gly Tyr Thr Ser Met Met Ser His Arg Ser Gly Glu Thr Glu 355360365 Asp Thr Thr Ile Ala Asp Leu Ala Val Ala Leu Asn Cys Gly Gln Ile 370375380 Lys Thr Gly Ala Pro Ala Arg Ser Asp Arg Val Ala Lys Tyr Asn Gln 385390395400 Leu Leu Arg Ile Glu Gln Leu Leu Gly Asp Ala Gly Val Tyr Ala Gly 405410415 Arg Ser Ala Phe Pro Arg Phe Gln Gly 420425 ...

Sequence Table <110>, Degussa - stock company in Wales <120> eno gene encoding nucleotide sequence of new <130> 990152 BT <140> <141> <160> 2 <170> Patent In Ver. 2.1 <210> 1 <211> 1578 <212> DNA <213> Corynebacterium glutamicum <220> <221> CDS <222> (151). . (1425) <400> 1 ggctggggat atgggtagtt ttcgccacta atttcaactg attgcctcat cgaaacaaga 60 ttcgtgcaac aattgggtgt agacgtgatt gaagacattt gatcacgtga ataattctag 120 ttagctccca agttggcata ggaggccaca gtg gct gaa atc ​​atg cac gta ttc 174 Val Ala Glu Ile Met His Val Phe 15 gct cgc gaa att ctc gac tcc cgc ggt aac cca acc gtc gag gca gag 222 Ala Arg Glu Ile Leu Asp Ser Arg Gly Asn Pro Thr Val Glu Ala Glu 101520 gtt ttc ctg gat gac ggt tcc cac ggt gtc gca ggt gtt cca tcc ggc 270 Val Phe Leu Asp Asp Gly Ser His Gly Val Ala Gly Val Pro Ser Gly 25303540 gca tcc acc ggc gtc cac gag gct cat gag ctg cgt gac ggt ggc gat 318 Ala Ser Thr Gly Val His Glu Ala His Glu Leu Arg Asp Gly Gly Asp 45 50 55 5 cgc tac ctg ggc aag ggc gtt ttg aag gca gtt gaa aac gtc aac gaa 366 Arg Tyr Leu Gly Lys Gly Val Leu Lys Ala Val Glu Asn Val Asn Glu 60 65 70 gaa atc ​​ggc gac gag ctc gct ggc cta gag gct gac gat cag cgc ctc 414 Glu Ile Gly Asp Glu Leu Ala Gly Leu Glu Ala Asp Asp Gln Arg Leu 75 80 85 atc gac gaa gca atg atc aag ctt gat ggc acc gcc aac aag tcc cgc 462 Ile Asp Glu Ala Met Ile Lys Leu Asp Gly Thr Ala Asn Lys Ser Arg 9095100 ctg ggt gca aac gca atc ​​ctt ggt gtt tcc atg gct gtt gca aag gct 510 Leu Gly Ala Asn Ala Ile Leu Gly Val Ser Met Ala Val Ala Lys Ala 105 110 115 120 gct gct gat tcc gca ggc ctc cca ctg ttc cgc tac atc ggt gga cca 558 Ala Ala Asp Ser Ala Gly Leu Pro Leu Phe Arg Tyr Ile Gly Gly Pro 125130135 aac gca cac gtt ctt cca gtt cca atg atg aac atc atc aac ggt ggc 606 Asn Ala His Val Leu Pro Val Pro Met Met Asn Ile Ile Asn Gly Gly 140 145 150 gct cac gct gac tcc ggt gtt gac gtt cag gaa ttc atg atc gct cca 654 Ala His Ala Asp Ser Gly Val Asp Val Gln Glu Phe Met Ile Ala Pro 155160165 atc ggt gca gag acc ttc tct gag gct ctc cgc aac ggc gcg gag gtc 702 Ile Gly Ala Glu Thr Phe Ser Glu Ala Leu Arg Asn Gly Ala Glu Val 170175180 tac cac gca ctg aag tcc gtc atc aag gaa aag ggc ctg tcc acc gga 750 Tyr His Ala Leu Lys Ser Val Ile Lys Glu Lys Gly Leu Ser Thr Gly 185190195200 ctt ggc gat gag ggc ggc ttc gct cct tcc gtc ggc tcc acc cgt gag 798 Leu Gly Asp Glu Gly Gly Phe Ala Pro Ser Val Gly Ser Thr Arg Glu 205210215 gct ctt gac ctt atc gtt gag gca atc ​​gag aag gct ggc ttc acc cca 846 Ala Leu Asp Leu Ile Val Glu Ala Ile Glu Lys Ala Gly Phe Thr Pro 220225230 ggc aag gac atc gct ctt gct ctg gac gtt gct tcc tct gag ttc ttc 894 Gly Lys Asp Ile Ala Leu Ala Leu Asp Val Ala Ser Ser Glu Phe Phe 235240245 aag gac ggc acc tac cac ttc gaa ggt ggc cag cac tcc gca gct gag 942 Lys Asp Gly Thr Tyr His Phe Glu Gly Gly Gln His Ser Ala Ala Glu 250 255 260 atg gca aac gtt tac gct gag ctc gtt gac gcg tac cca atc ​​gtc tcc 990 Met Ala Asn Val Tyr Ala Glu Leu Val Asp Ala Tyr Pro Ile Val Ser 265270275280 atc gag gac cca ctg cag gaa gat gac tgg gag ggt tac acc aac ctc 1038 Ile Glu Asp Pro Leu Gln Glu Asp Asp Trp Glu Gly Tyr Thr Asn Leu 285290295 acc gca acc atc ggc gac aag gtt cag atc gtt ggc gac gac ttc ttc 1086 Thr Ala Thr Ile Gly Asp Lys Val Gln Ile Val Gly Asp Asp Phe Phe 300305310 gtc acc aac cct gag cgc ctg aag gag ggc atc gct aag aag gct gcc 1134 Val Thr Asn Pro Glu Arg Leu Lys Glu Gly Ile Ala Lys Lys Ala Ala 315320325 aac tcc atc ctg gtt aag gtg aac cag atc ggt acc ctc acc gag acc 1182 Asn Ser Ile Leu Val Lys Val Asn Gln Ile Gly Thr Leu Thr Glu Thr 330335340 ttc gac gct gtc gac atg gct cac cgc gca ggc tac acc tcc atg atg 1230 Phe Asp Ala Val Asp Met Ala His Arg Ala Gly Tyr Thr Ser Met Met 345350355360 tcc cac cgt tcc ggt gag acc gag gac acc acc att gct gac ctc gca 1278 Ser His Arg Ser Gly Glu Thr Glu Asp Thr Thr Ile Ala Asp Leu Ala 365370375 gtt gca ctc aac tgt ggc cag atc aag act ggt gct cca gca cgt tcc 1326 Val Ala Leu Asn Cys Gly Gln Ile Lys Thr Gly Ala Pro Ala Arg Ser 380385390 gac cgt gtc gca aag tac aac cag ctt ctc cgc atc gag cag ctg ctt 1374 Asp Arg Val Ala Lys Tyr Asn Gln Leu Leu Arg Ile Glu Gln Leu Leu 395400405 ggc gac gcc ggc gtc tac gca ggt cgc agc gca ttc cca cgc ttt cag 1422 Gly Asp Ala Gly Val Tyr Ala GIy Arg Ser Ala Phe Pro Arg Phe Gln 410415420 ggc taaataaaag cgcttttcga cgcccggtaa cctcaaggtt gccgggcgtc 1475 Gly 425 gttgccttac tactgttact ggtgtgacta tgatcgagga ttatggcaaa gcagaagaaa 1535 actcataaag gccttgttcc tgtctcaagc agggaacgtg ctt 1578 <210> 2 <211> 425 <212> PRT <213> Corynebacterium glutamicum <400> 2 Val Ala Glu Ile Met His Val Phe Ala Arg Glu Ile Leu Asp Ser Arg 151015 Gly Asn Pro Thr Val Glu Ala Glu Val Phe Leu Asp Asp Gly Ser His 202530 Gly Val Ala Gly Val Pro Ser Gly Ala Ser Thr Gly Val His Glu Ala 35 40 45 His Glu Leu Arg Asp Gly Gly Asp Arg Tyr Leu Gly Lys Gly Val Leu 50 55 60 Lys Ala Val Glu Asn Val Asn Glu Glu Ile Gly Asp Glu Leu Ala Gly 65 70 75 80 Leu Glu Ala Asp Asp Gln Arg Leu Ile Asp Glu Ala Met Ile Lys Leu 85 90 95 Asp Gly Thr Ala Asn Lys Ser Arg Leu Gly Ala Asn Ala Ile Leu Gly 100 105 110 Val Ser Met Ala Val Ala Lys Ala Ala Ala Asp Ser Ala Gly Leu Pro 115120125 Leu Phe Arg Tyr Ile Gly Gly Pro Asn Ala His Val Leu Pro Val Pro 130135140 Met Met Asn Ile Ile Asn Gly Gly Ala His Ala Asp Ser Gly Val Asp 145150155160 Val Gln Glu Phe Met Ile Ala Pro Ile Gly Ala Glu Thr Phe Ser Glu 165170175 Ala Leu Arg Asn Gly Ala Glu Val Tyr His Ala Leu Lys Ser Val Ile 180185190 Lys Glu Lys Gly Leu Ser Thr Gly Leu Gly Asp Glu Gly Gly Phe Ala 195200205 Pro Ser Val Gly Ser Thr Arg Glu Ala Leu Asp Leu Ile Val Glu Ala 210215220 Ile Glu Lys Ala Gly Phe Thr Pro Gly Lys Asp Ile Ala Leu Ala Leu 225230235240 Asp Val Ala Ser Ser Glu Phe Phe Lys Asp Gly Thr Tyr His Phe Glu 245250255 Gly Gly Gln His Ser Ala Ala Glu Met Ala Asn Val Tyr Ala Glu Leu 260265270 Val Asp Ala Tyr Pro Ile Val Ser Ile Glu Asp Pro Leu Gln Glu Asp 275280285 Asp Trp Glu Gly Tyr Thr Asn Leu Thr Ala Thr Ile Gly Asp Lys Val 290295300 Gln Ile Val Gly Asp Asp Phe Phe Val Thr Asn Pro Glu Arg Leu Lys 305310315320 Glu Gly Ile Ala Lys Lys Ala Ala Asn Ser Ile Leu Val Lys Val Asn 325330335 Gln Ile Gly Thr Leu Thr Glu Thr Phe Asp Ala Val Asp Met Ala His 340345350 Arg Ala Gly Tyr Thr Ser Met Met Ser His Arg Ser Gly Glu Thr Glu 355360365 Asp Thr Thr Ile Ala Asp Leu Ala Val Ala Leu Asn Cys Gly Gln Ile 370375380 Lys Thr Gly Ala Pro Ala Arg Ser Asp Arg Val Ala Lys Tyr Asn Gln 385390395400 Leu Leu Arg Ile Glu Gln Leu Leu Gly Asp Ala Gly Val Tyr Ala Gly 405410415 Arg Ser Ala Phe Pro Arg Phe Gln Gly 420425 ...

Claims (26)

1. the desacetoxy cephalosporin synthase (DAOCS) of structure that has the X ray coordinate definition of this paper structure A or structure B.

2. and metal, for example iron or the plumbous DAOCS that forms the complex body form, and optional can not select in the presence of substrate and/or substrate analogue or inhibitor, have structure by the definition of this paper X ray coordinate.

3. the DAOCS of claim 2, wherein substrate is a penicillin N, penicillin G, 2-oxoglutaric acid or dioxygen, and inhibitor is selected from the acid of N-oxamido-, pyridine carboxylic acid class and nitrous oxide.

4.DAOCS the application of three-dimensional structure in the modification of DAOCS or other relevant 2-oxoglutaric acid dependent enzyme.

5. the described application of claim 4, wherein Xiang Guan 2-oxoglutaric acid dependent enzyme is DACS, the oxygenase of cephamycin C is introduced 7 α-methoxyl group in DAOC/DACS or participation.

6. claim 5 is used, and is used for DAOCS, and DACS or DAOC/DACS modify, so that they more effectively accept non-natural substrates than wild-type enzyme.

7. claim 5 is used, be used for DAOCS, and DACS, DAOC/DACS modifies, so that they change natural substrate in medicine or medication preparation useful as intermediates.

8. application as claimed in claim 6, wherein non-natural substrates is a penicillin, comprises penicillin G, penicillin v, 6-amino-penicillanic acid, amoxycilline Trihydrate bp, or the penicillins of tool phenylglycocoll or glycin side chain.

9. the application of claim 6, wherein non-natural substrates is a cynnematin.

10. the application of claim 6, wherein non-natural substrates is a seed amino acid, comprises proteinogen amino acid, or a kind of peptide.

11. each described application of claim 6-8, penicillin G wherein, penicillin v, other non-natural substrates or penicillin N are converted into cynnematin or exomethylenecepham rhzomorph.

12. one kind has as herein defined the significantly enzyme of sequence similarity with DAOCS, wherein modified and at least one amino-acid residue at least one following site of the side chain binding site of penicillin N or DAOC changes the another one amino-acid residue into or is lacked:

Thr72, Arg74, Arg75, Glu156, Leu158, Arg160, Arg162, Leu186, Ser187, Phe225, Phe264, Arg266, Asp301, Tyr302, Val303, Asn304; And/or at least one extra amino-acid residue is inserted into regional 300-311; Prerequisite is and may also changes with interactional other residue acid of upper amino acid to change one of above cooperating.

13. one kind has the enzyme of remarkable sequence similarity as defined herein with DAOCS, wherein the penicillin of penicillin N or DAOC/cephamycin binding site is through modifying, and at least one following amino-acid residue is changed or is lacked: Ile88, Arg160, Arg162, Phe164, Met180, Thr190, Ile192, Phe225, Pro241, Val245, Val262, Phe264, Ile305, Arg306, Arg307, and/or at least one additional amino acid residue is inserted in regional 300-311; Prerequisite is and also changes with interactional other residual other residue of upper amino acid with the change one of above cooperating.

14. the enzyme of claim 12 or claim 13, it is the mutant of DAOCS or DACS or DAOC/DACS.

15. each described enzyme among the claim 12-14, wherein the side chain of penicillin/cynnematin of penicillin N or DAOC and binding site through modification and claim 12 and 13 defined in residue at least a change or lack.

16. each described enzyme of claim 12-15 is wherein introduced two or more complementary sudden changes to produce or the disappearance binding interactions, comprises hydrogen bond, static, or hydrophobic interaction.

17. each the gene of enzyme of coding claim 12-16.

18. under fermentation conditions can express the microorganism of the gene of claim 17.

19. the application of the microorganism of claim 18 in the production of the beta-lactam of penicillin or cynnematin (comprising cephem (cepham)) family.

20. the described application of claim 19; wherein microorganism contains other modified enzyme in penicillin and the cynnematin biosynthetic pathway; comprise the plain N synthase of different blue or green enzyme, hydroamidase/Transacetylase, or L-δ-(amino adipyl)-L-halfcystine-D-Xie Ansuan (ACV) synthase.

21. method, comprise enzyme that the three-dimensional structure of using DAOCS is determined or prediction is relevant in addition 2-oxoglutaric acid relies on or from the structure of the relevant enzyme of non-penicillin and cynnematin biosynthetic pathway, and the structural information that obtains of utilization is modified other enzyme or is designed the inhibitor of other enzyme.

22. the method for claim 21, enzyme or involved enzyme that wherein said other relevant 2-oxoglutaric acid relies on are 1-amino-cyclopropane-1-carboxylate oxydase, Plant hormones regulators,gibberellins C-20 oxydase, flavone synthase, flavanone 3 B-hydroxylases, l-tropine tropate 6 B-hydroxylases, prolyl 4-hydroxylase, prolyl 3-hydroxylase, aspartoyl hydroxylase, lysyl hydroxylase, Protocollagen prolyl hydroxylase, gamma-butyrobetaine hydroxylase, the enzyme in the Herbicid resistant mechanism, excellent alkanoic acid (Clavaminate) synthase, participate in the biosynthetic oxygenase of carbapenem, ethene from pseudomonas syringae forms enzyme, to the oxygenase of phytol oxidation in hydroxyphenyl pyruvic acid dioxygenase and the participant liver peroxysome.

23. the method in claim 21 or 22, other enzyme described in the spring by disappearance, are added or are changed and modify; Modification occurs in one or more site of claim 12 or 13 definition; Or using following information design inhibitor: Asp185, His183 and His243 are as the part of iron; Arg258 and Ser260 and Fe combine with 2-oxoglutaric acid; Met180, Phe225, Leu31 and Val245 are near the iron binding site; Tyr33, Arg160, Arg162, Phe164, Ile192, Gln194, Leu204, Leu223, Leu215 is for very important with the structure of some part of 2-oxoglutaric acid bonded avtive spot; Arg160 and Arg162 are for combining very important with amino acid or peptide derived substrates.

24. each described method of claim 21-23, wherein said other enzyme is a prolyl 4-hydroxylase, prolyl 3-hydroxylase, aspartoyl hydroxylase, or lysyl hydroxylase, and inhibitor is applied to the human disease is comprised in fiber disease liver cirrhosis and the arthritic treatment.

25. each described method of claim 21-23, wherein said other relevant enzyme are to methylolacetone acid dioxygenase, and inhibitor is applied in the treatment to certain inherited disease.

26. each described method of claim 21-23, wherein said other enzyme participates in Herbicid resistant and its information is used to design novel herbicide to overcome resistance problem.

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