EzCatDB: M00309
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DB codeM00309
RLCP classification1.13.30010.30 : Hydrolysis
CATH domainDomain 11.10.439.10 : Penicillin Amidohydrolase; domain 1
Domain 23.60.20.10 : Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1Catalytic domain
Domain 32.30.120.10 : Penicillin G acylase, beta-roll domain
Domain 41.10.1400.10 : Penicillin amidase (Acylase) alpha subunit, N-terminal domain
E.C.3.5.1.93
MACiEM0288


Enzyme Name
UniProtKBKEGG

Q9L5D6P07662O86089
Protein nameGlutaryl-7-aminocephalosporanic-acid acylaseGlutaryl-7-aminocephalosporanic-acid acylase
Glutaryl-7-aminocephalosporanic-acid acylase
7Beta-(4-carboxybutanamido)cephalosporanic acid acylase
Cephalosporin C acylase
Glutaryl-7-ACA acylase
CA
GCA
GA
Cephalosporin acylase
Glutaryl-7-aminocephalosporanic acid acylase
GL-7-ACA acylase
SynonymsGlutaryl-7-ACA acylase
EC 3.5.1.93
7-beta-(4-carboxybutanamido)cephalosporanic acid acylase
CAD
GL-7-ACA acylase
GCA
Glutaryl-7-ACA acylase
EC 3.5.1.93
7-beta-(4-carboxybutanamido)cephalosporanic acid acylase
GL-7-ACA acylase
GCA
Cephalosporin acylase
ContainsGlutaryl-7-aminocephalosporanic-acid acylase subunit alpha
(Glutaryl-7-ACA acylase subunit alpha)
Glutaryl-7-aminocephalosporanic-acid acylase subunit beta
(Glutaryl-7-ACA acylase subunit beta)
Glutaryl-7-aminocephalosporanic-acid acylase subunit alpha
(Glutaryl-7-ACA acylase subunit alpha)
Glutaryl-7-aminocephalosporanic-acid acylase subunit beta
(Glutaryl-7-ACA acylase subunit beta)
None
MEROPSS45.002 (Serine)
S45.002 (Serine)
S45.002 (Serine)
PfamPF01804 (Penicil_amidase)
[Graphical view]
PF01804 (Penicil_amidase)
[Graphical view]
PF01804 (Penicil_amidase)
[Graphical view]

KEGG pathways
MAP codePathways
MAP00311Penicillin and cephalosporin biosynthesis

UniProtKB:Accession NumberQ9L5D6P07662O86089
Entry nameG7AC_BREDIG7AC_PSEU7O86089_9PROT
Activity(7R)-7-(4-carboxybutanamido)cephalosporanate + H(2)O = (7R)-7-aminocephalosporanate + glutarate.(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O = (7R)-7-aminocephalosporanate + glutarate.
SubunitHeterodimer of a small subunit and a large subunit processed from the same precursor.Heterotetramer of two alpha and two beta subunits processed from the same precursor.
Subcellular locationPeriplasm (Potential).Periplasm
Cofactor



Compound table: links to PDB-related databases & PoSSuM

SubstratesProductsintermediates
KEGG-idC15666C00001C07756C00489I00190I00191I00192
Compound(7R)-7-(4-carboxybutanamido)cephalosporanateH2O(7R)-7-aminocephalosporanateglutaratePeptidyl-Ser-7-glutarylaminocephalosporanate (tetrahedral transition-state)Peptidyl-Ser-glutarate (acyl-enzyme intermediate)Peptidyl-Ser-glutarate (tetrahedral transition-state)
Typeamide group,carbohydrate,carboxyl group,sulfide groupH2Oamide group,amine group,carbohydrate,carboxyl group,sulfide groupcarboxyl group


ChEBI
15377





PubChem
962
22247451





               
1kehA02Unbound UnboundUnboundUnboundUnboundUnbound
1fm2A00Unbound UnboundUnboundUnboundUnboundUnbound
1jvzA00Unbound UnboundUnboundUnboundUnboundUnbound
1jw0A00Unbound UnboundUnboundUnboundUnboundUnbound
3s8rA02Unbound UnboundUnboundUnboundUnboundUnbound
3s8rB02Unbound UnboundUnboundUnboundUnboundUnbound
1or0A00Unbound UnboundUnboundUnboundUnboundUnbound
1or0C00Unbound UnboundUnboundUnboundUnboundUnbound
1gk0A00Unbound UnboundUnboundUnboundUnboundUnbound
1gk0C00Unbound UnboundUnboundUnboundUnboundUnbound
1gk1A00Unbound UnboundUnboundUnboundUnboundUnbound
1gk1C00Unbound UnboundUnboundUnboundUnboundUnbound
1ghdA00Unbound UnboundUnboundUnboundUnboundUnbound
2ae3A00Unbound UnboundUnboundUnboundUnboundUnbound
2ae4A00Unbound UnboundUnboundUnboundUnboundUnbound
2ae5A00Unbound UnboundUnboundUnboundUnboundUnbound
1kehA01Unbound UnboundUnboundUnboundUnboundUnbound
1fm2B01Unbound UnboundUnboundUnboundUnboundUnbound
1jvzB01Bound:CEN UnboundUnboundUnboundUnboundUnbound
1jw0B01Unbound UnboundBound:GUAUnboundUnboundUnbound
3s8rA01Unbound UnboundUnboundUnboundUnboundUnbound
3s8rB01Unbound UnboundUnboundUnboundUnboundUnbound
1or0B01Unbound UnboundUnboundUnboundUnboundUnbound
1or0D01Unbound UnboundUnboundUnboundUnboundUnbound
1gk0B01Unbound UnboundUnboundUnboundUnboundTransition-state-analogue:PO4-EDO
1gk0D01Unbound UnboundUnboundUnboundUnboundTransition-state-analogue:PO4-EDO
1gk1B01Unbound UnboundUnboundUnboundUnboundUnbound
1gk1D01Unbound UnboundUnboundUnboundUnboundUnbound
1ghdB01Unbound UnboundUnboundUnboundUnboundUnbound
2ae3B01Unbound UnboundUnboundUnboundUnboundUnbound
2ae4B01Unbound UnboundUnboundUnboundUnboundUnbound
2ae5B01Unbound UnboundUnboundUnboundUnboundUnbound
1kehA03Unbound UnboundUnboundUnboundUnboundUnbound
1fm2B03Unbound UnboundUnboundUnboundUnboundUnbound
1jvzB03Unbound UnboundUnboundUnboundUnboundUnbound
1jw0B03Unbound UnboundUnboundUnboundUnboundUnbound
3s8rA03Unbound UnboundUnboundUnboundUnboundUnbound
3s8rB03Unbound UnboundUnboundUnboundUnboundUnbound
1or0B03Unbound UnboundUnboundUnboundUnboundUnbound
1or0D03Unbound UnboundUnboundUnboundUnboundUnbound
1gk0B03Unbound UnboundUnboundUnboundUnboundUnbound
1gk0D03Unbound UnboundUnboundUnboundUnboundUnbound
1gk1B03Unbound UnboundUnboundUnboundUnboundUnbound
1gk1D03Unbound UnboundUnboundUnboundUnboundUnbound
1ghdB03Unbound UnboundUnboundUnboundUnboundUnbound
2ae3B02Unbound UnboundUnboundUnboundUnboundUnbound
2ae4B02Unbound UnboundUnboundUnboundUnboundUnbound
2ae5B02Unbound UnboundUnboundUnboundUnboundUnbound
1kehA04Unbound UnboundUnboundUnboundUnboundUnbound
1fm2B02Unbound UnboundUnboundUnboundUnboundUnbound
1jvzB02Unbound UnboundUnboundUnboundUnboundUnbound
1jw0B02Unbound UnboundUnboundUnboundUnboundUnbound
3s8rA04Unbound UnboundUnboundUnboundUnboundUnbound
3s8rB04Unbound UnboundUnboundUnboundUnboundUnbound
1or0B02Unbound UnboundUnboundUnboundUnboundUnbound
1or0D02Unbound UnboundUnboundUnboundUnboundUnbound
1gk0B02Unbound UnboundUnboundUnboundUnboundUnbound
1gk0D02Unbound UnboundUnboundUnboundUnboundUnbound
1gk1B02Unbound UnboundUnboundUnboundUnboundUnbound
1gk1D02Unbound UnboundUnboundUnboundUnboundUnbound
1ghdB02Unbound UnboundUnboundUnboundUnboundUnbound
2ae3B03Unbound UnboundUnboundUnboundUnboundUnbound
2ae4B03Unbound UnboundUnboundUnboundUnboundUnbound
2ae5B03Unbound UnboundUnboundUnboundUnboundUnbound

Active-site residues
pdbCatalytic residuesMain-chain involved in catalysiscomment
           
1kehA02       
 
 
1fm2A00       
 
 
1jvzA00       
 
 
1jw0A00       
 
 
3s8rA02       
 
 
3s8rB02       
 
 
1or0A00       
 
 
1or0C00       
 
 
1gk0A00       
 
 
1gk0C00       
 
 
1gk1A00       
 
 
1gk1C00       
 
 
1ghdA00       
 
 
2ae3A00       
 
 
2ae4A00       
 
 
2ae5A00       
 
 
1kehA01       ;HIS 192;ASN 413
       ;HIS 192;VAL 239
mutant S170A
1fm2B01SER 170;HIS 192;ASN 413
SER 170;HIS 192;VAL 239
 
1jvzB01SER 170;HIS 192;ASN 413
SER 170;HIS 192;VAL 239
 
1jw0B01SER 170;HIS 192;ASN 413
SER 170;HIS 192;VAL 239
 
3s8rA01       ;HIS 192;ASN 413
       ;HIS 192;VAL 239
mutant S170A
3s8rB01       ;HIS 192;ASN 413
       ;HIS 192;VAL 239
mutant S170A
1or0B01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
1or0D01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
1gk0B01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
1gk0D01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
1gk1B01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
1gk1D01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
1ghdB01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
2ae3B01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
2ae4B01SER   1;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
 
2ae5B01       ;HIS  23;ASN 244
SER   1;HIS  23;VAL  70
mutant S1C
1kehA03       
 
 
1fm2B03       
 
 
1jvzB03       
 
 
1jw0B03       
 
 
3s8rA03       
 
 
3s8rB03       
 
 
1or0B03       
 
 
1or0D03       
 
 
1gk0B03       
 
 
1gk0D03       
 
 
1gk1B03       
 
 
1gk1D03       
 
 
1ghdB03       
 
 
2ae3B02       
 
 
2ae4B02       
 
 
2ae5B02       
 
 
1kehA04       
 
 
1fm2B02       
 
 
1jvzB02       
 
 
1jw0B02       
 
 
3s8rA04       
 
 
3s8rB04       
 
 
1or0B02       
 
 
1or0D02       
 
 
1gk0B02       
 
 
1gk0D02       
 
 
1gk1B02       
 
 
1gk1D02       
 
 
1ghdB02       
 
 
2ae3B03       
 
 
2ae4B03       
 
 
2ae5B03       
 
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[4]p.1063
[10]p.95-97

references
[1]
PubMed ID11099866
JournalBiochim Biophys Acta
Year2000
Volume1523
Pages123-7
AuthorsLee YS, Kim HW, Lee KB, Park SS
TitleInvolvement of arginine and tryptophan residues in catalytic activity of glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16.
[2]
PubMed ID10991936
JournalJ Biol Chem
Year2000
Volume275
Pages39200-6
AuthorsLee YS, Kim HW, Park SS
TitleThe role of alpha-amino group of the N-terminal serine of beta subunit for enzyme catalysis and autoproteolytic activation of glutaryl 7-aminocephalosporanic acid acylase.
[3]
PubMed ID10945972
JournalJ Struct Biol
Year2000
Volume131
Pages79-81
AuthorsKwon TH, Rhee S, Lee YS, Park SS, Kim KH
TitleCrystallization and preliminary X-Ray diffraction analysis of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp. GK16.
[4]
PubMed ID11080627
JournalStructure
Year2000
Volume8
Pages1059-68
AuthorsKim Y, Yoon K, Khang Y, Turley S, Hol WG
TitleThe 2.0 A crystal structure of cephalosporin acylase.
Related PDB1fm2
[5]
PubMed ID11755403
JournalChem Biol
Year2001
Volume8
Pages1253-64
AuthorsKim Y, Hol WG
TitleStructure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: insight into the basis of its substrate specificity.
Related PDB1jvz,1jw0
[6]
PubMed ID11604409
JournalJ Biol Chem
Year2001
Volume276
Pages48376-81
AuthorsKim S, Kim Y
TitleActive site residues of cephalosporin acylase are critical not only for enzymatic catalysis but also for post-translational modification.
[7]
PubMed ID11782466
JournalJ Biol Chem
Year2002
Volume277
Pages10256-64
AuthorsHuang X, Zeng R, Ding X, Mao X, Ding Y, Rao Z, Xie Y, Jiang W, Zhao G
TitleAffinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.
[8]
PubMed ID11706000
JournalJ Biol Chem
Year2002
Volume277
Pages2823-9
AuthorsKim Y, Kim S, Earnest TN, Hol WG
TitlePrecursor structure of cephalosporin acylase. Insights into autoproteolytic activation in a new N-terminal hydrolase family.
Related PDB1keh
[9]
PubMed ID12198140
JournalJ Biol Chem
Year2002
Volume277
Pages42121-7
AuthorsOtten LG, Sio CF, Vrielink J, Cool RH, Quax WJ
TitleAltering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit.
[10]
PubMed ID11742126
JournalProtein Sci
Year2002
Volume11
Pages92-103
AuthorsFritz-Wolf K, Koller KP, Lange G, Liesum A, Sauber K, Schreuder H, Aretz W, Kabsch W
TitleStructure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C.
Related PDB1gk1,1gk0
[11]
PubMed ID14511642
JournalBiochem Biophys Res Commun
Year2003
Volume310
Pages19-27
AuthorsOh B, Kim M, Yoon J, Chung K, Shin Y, Lee D, Kim Y
TitleDeacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues.
[12]
PubMed ID14680829
JournalBiochem Biophys Res Commun
Year2003
Volume312
Pages755-60
AuthorsSio CF, Otten LG, Cool RH, Quax WJ
TitleAnalysis of a substrate specificity switch residue of cephalosporin acylase.
[13]
PubMed ID12680762
JournalBiochemistry
Year2003
Volume42
Pages4084-93
AuthorsKim JK, Yang IS, Rhee S, Dauter Z, Lee YS, Park SS, Kim KH
TitleCrystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation.
Related PDB3s8r,1or0
[14]
PubMed ID15174165
JournalChembiochem
Year2004
Volume5
Pages820-5
AuthorsOtten LG, Sio CF, van der Sloot AM, Cool RH, Quax WJ
TitleMutational analysis of a key residue in the substrate specificity of a cephalosporin acylase.
[15]
PubMed ID15200051
JournalProtein J
Year2004
Volume23
Pages197-204
AuthorsMao X, Wang W, Jiang W, Zhao GP
TitleHis23beta and Glu455beta of the Pseudomonas sp. 130 glutaryl-7-amino cephalosporanic acid acylase are crucially important for efficient autoproteolysis and enzymatic catalysis.
[16]
PubMed ID16446446
JournalProc Natl Acad Sci U S A
Year2006
Volume103
Pages1732-7
AuthorsKim JK, Yang IS, Shin HJ, Cho KJ, Ryu EK, Kim SH, Park SS, Kim KH
TitleInsight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries.
Related PDB2adv,2ae3,2ae4,2ae5
[17]
PubMed ID19800869
JournalBiochem Biophys Res Commun
Year2009
Volume390
Pages342-8
AuthorsCho KJ, Kim JK, Lee JH, Shin HJ, Park SS, Kim KH
TitleStructural features of cephalosporin acylase reveal the basis of autocatalytic activation.
[18]
PubMed ID20606279
JournalActa Crystallogr Sect F Struct Biol Cryst Commun
Year2010
Volume66
Pages808-10
AuthorsAnandan A, Vallet C, Coyle T, Moustafa IM, Vrielink A
TitleCrystallization and preliminary diffraction analysis of an engineered cephalosporin acylase.
[19]
PubMed ID21576250
JournalJ Biol Chem
Year2011
Volume286
Pages24476-86
AuthorsYin J, Deng Z, Zhao G, Huang X
TitleThe N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis.

comments
This enzyme belongs to the Ntn hydrolase family (CATH 3.60.20.10), whose N-terminal residue plays a catalytic residue (see [2]). Moreover, this enzyme belongs to S45 peptidase family (see [19]).
The precursor protein of this enzyme is composed of signal peptide, alpha-subunit, beta-subunit and spaceer-sequence, which is between alpha-subunit and beta-subunit regions ([4], [8], [10], [13], [14], [15], [16], [19]). This precursor protein is activated by a two-step autoproteolysis ([2], [8], [13], [14], [15], [16], [19]).
The first step of the autoproteolytic activation is an intramolecular cleavage of the precursor protein between Gly169 and Ser170 to release alpha'-subunit, which contains spacer sequence, and beta-subunit ([8], [13], [14], [15], [16], [19]). At this step, Ser170 play a nucleophilic role, to attack on the carbonyl carbon of Gly169 (see [8], [13], [16], [19]). This nucleophilic attack leads to formation of tetrahedral transition-state that is stabilized by Asn413 and an oxyanion hole of His192 and Val239 ([13]).
The second step is a cleavage of the alpha'-subunit between Gly160 and Asp161, to produce alpha-subunit and the spacer peptide ([8], [13], [14], [15], [16], [19]). Initially, the second cleavage is thought to proceed by intermolecular interaction (see [2], [6], [8], [12]). More recently, it is reported that the second cleavage step is an intramolecular reaction, in which Ser170 (or Ser1 of beta subunit) plays a nucleophicil role ([16], [19]).
The catalytic reaction of this enzyme may proceed as follows ([4], [10]):
(0) The sidechain of His23-beta (His192 of 1fm2B) maintains the uncharged state of the alpha-amino group of Ser1-beta (Ser170). Here, Glu455-beta may modulate the activity of His23-beta. His23-beta and Glu455-beta form a catalytic dyad ([10]).
(1) The alpha-amino group of Ser1-beta (Ser170) acts as a general base, to activate the sidechain hydroxyl group of Ser1-beta through a water molecule.
(2) Ser1-beta makes a nucleophilic attack on the acyl group of the substrate, leading to a tetrahedral transition-state (I00190).
(3) The negatively charged transition-state is stabilized by an oxyanion hole formed by two amide groups; Mainchain amide groups of His23-beta (His192) and Val70-beta (Val239) and sidechain amide of Asn244-beta (Asn413).
(4) (The amino group of Ser1-beta may act as a general acid to protonate the tetrahedral transition-state, which will lead to collapse of the tetrahedral transition-state.) The collapse of the tetrahedral transition-state leads to a seryl acyl-enzyme intermediate (I00191), releasing 7-aminochephalosporanate (C07756).
(5) Another water molecule, which may be activated by the amino group of Ser1-beta, attacks on the acyl-enzyme intermediate (I00191), leading to another tetrahedral transition-state (I00192). This transition-state (I00192) is also stabilized by the oxyanion hole.
(6) Finally, this transition-state collapses, releasing free glutarate.

createdupdated
2013-02-252016-01-27


Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
Supported by the commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) (October 2012 - )
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