DB code: M00309

RLCP classification 1.13.30010.30 : Hydrolysis
CATH domain 1.10.439.10 : Penicillin Amidohydrolase; domain 1
3.60.20.10 : Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 Catalytic domain
2.30.120.10 : Penicillin G acylase, beta-roll domain
1.10.1400.10 : Penicillin amidase (Acylase) alpha subunit, N-terminal domain
E.C. 3.5.1.93
CSA
M-CSA
MACiE M0288

CATH domain Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Contains MEROPS Pfam
Q9L5D6 Glutaryl-7-aminocephalosporanic-acid acylase
Glutaryl-7-ACA acylase
EC 3.5.1.93
7-beta-(4-carboxybutanamido)cephalosporanic acid acylase
CAD
GL-7-ACA acylase
GCA
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)
S45.002 (Serine)
PF01804 (Penicil_amidase)
[Graphical View]
P07662 Glutaryl-7-aminocephalosporanic-acid acylase
Glutaryl-7-ACA acylase
EC 3.5.1.93
7-beta-(4-carboxybutanamido)cephalosporanic acid acylase
GL-7-ACA acylase
GCA
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)
S45.002 (Serine)
PF01804 (Penicil_amidase)
[Graphical View]
O86089
Cephalosporin acylase
None S45.002 (Serine)
PF01804 (Penicil_amidase)
[Graphical View]

KEGG enzyme name
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

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
Q9L5D6 G7AC_BREDI (7R)-7-(4-carboxybutanamido)cephalosporanate + H(2)O = (7R)-7-aminocephalosporanate + glutarate. Heterodimer of a small subunit and a large subunit processed from the same precursor. Periplasm (Potential).
P07662 G7AC_PSEU7 (7R)-7-(4-carboxybutanamido)cephalosporanate + H2O = (7R)-7-aminocephalosporanate + glutarate. Heterotetramer of two alpha and two beta subunits processed from the same precursor. Periplasm
O86089 O86089_9PROT

KEGG Pathways
Map code Pathways E.C.
MAP00311 Penicillin and cephalosporin biosynthesis

Compound table
Substrates Products Intermediates
KEGG-id C15666 C00001 C07756 C00489 I00190 I00191 I00192
E.C.
Compound (7R)-7-(4-carboxybutanamido)cephalosporanate H2O (7R)-7-aminocephalosporanate glutarate Peptidyl-Ser-7-glutarylaminocephalosporanate (tetrahedral transition-state) Peptidyl-Ser-glutarate (acyl-enzyme intermediate) Peptidyl-Ser-glutarate (tetrahedral transition-state)
Type amide group,carbohydrate,carboxyl group,sulfide group H2O amide group,amine group,carbohydrate,carboxyl group,sulfide group carboxyl group
ChEBI 15377
 15377
PubChem 22247451
 962
 22247451
 962
1kehA02 Unbound Unbound Unbound Unbound Unbound Unbound
1fm2A00 Unbound Unbound Unbound Unbound Unbound Unbound
1jvzA00 Unbound Unbound Unbound Unbound Unbound Unbound
1jw0A00 Unbound Unbound Unbound Unbound Unbound Unbound
3s8rA02 Unbound Unbound Unbound Unbound Unbound Unbound
3s8rB02 Unbound Unbound Unbound Unbound Unbound Unbound
1or0A00 Unbound Unbound Unbound Unbound Unbound Unbound
1or0C00 Unbound Unbound Unbound Unbound Unbound Unbound
1gk0A00 Unbound Unbound Unbound Unbound Unbound Unbound
1gk0C00 Unbound Unbound Unbound Unbound Unbound Unbound
1gk1A00 Unbound Unbound Unbound Unbound Unbound Unbound
1gk1C00 Unbound Unbound Unbound Unbound Unbound Unbound
1ghdA00 Unbound Unbound Unbound Unbound Unbound Unbound
2ae3A00 Unbound Unbound Unbound Unbound Unbound Unbound
2ae4A00 Unbound Unbound Unbound Unbound Unbound Unbound
2ae5A00 Unbound Unbound Unbound Unbound Unbound Unbound
1kehA01 Unbound Unbound Unbound Unbound Unbound Unbound
1fm2B01 Unbound Unbound Unbound Unbound Unbound Unbound
1jvzB01 Bound:CEN Unbound Unbound Unbound Unbound Unbound
1jw0B01 Unbound Unbound Bound:GUA Unbound Unbound Unbound
3s8rA01 Unbound Unbound Unbound Unbound Unbound Unbound
3s8rB01 Unbound Unbound Unbound Unbound Unbound Unbound
1or0B01 Unbound Unbound Unbound Unbound Unbound Unbound
1or0D01 Unbound Unbound Unbound Unbound Unbound Unbound
1gk0B01 Unbound Unbound Unbound Unbound Unbound Transition-state-analogue:PO4-EDO
1gk0D01 Unbound Unbound Unbound Unbound Unbound Transition-state-analogue:PO4-EDO
1gk1B01 Unbound Unbound Unbound Unbound Unbound Unbound
1gk1D01 Unbound Unbound Unbound Unbound Unbound Unbound
1ghdB01 Unbound Unbound Unbound Unbound Unbound Unbound
2ae3B01 Unbound Unbound Unbound Unbound Unbound Unbound
2ae4B01 Unbound Unbound Unbound Unbound Unbound Unbound
2ae5B01 Unbound Unbound Unbound Unbound Unbound Unbound
1kehA03 Unbound Unbound Unbound Unbound Unbound Unbound
1fm2B03 Unbound Unbound Unbound Unbound Unbound Unbound
1jvzB03 Unbound Unbound Unbound Unbound Unbound Unbound
1jw0B03 Unbound Unbound Unbound Unbound Unbound Unbound
3s8rA03 Unbound Unbound Unbound Unbound Unbound Unbound
3s8rB03 Unbound Unbound Unbound Unbound Unbound Unbound
1or0B03 Unbound Unbound Unbound Unbound Unbound Unbound
1or0D03 Unbound Unbound Unbound Unbound Unbound Unbound
1gk0B03 Unbound Unbound Unbound Unbound Unbound Unbound
1gk0D03 Unbound Unbound Unbound Unbound Unbound Unbound
1gk1B03 Unbound Unbound Unbound Unbound Unbound Unbound
1gk1D03 Unbound Unbound Unbound Unbound Unbound Unbound
1ghdB03 Unbound Unbound Unbound Unbound Unbound Unbound
2ae3B02 Unbound Unbound Unbound Unbound Unbound Unbound
2ae4B02 Unbound Unbound Unbound Unbound Unbound Unbound
2ae5B02 Unbound Unbound Unbound Unbound Unbound Unbound
1kehA04 Unbound Unbound Unbound Unbound Unbound Unbound
1fm2B02 Unbound Unbound Unbound Unbound Unbound Unbound
1jvzB02 Unbound Unbound Unbound Unbound Unbound Unbound
1jw0B02 Unbound Unbound Unbound Unbound Unbound Unbound
3s8rA04 Unbound Unbound Unbound Unbound Unbound Unbound
3s8rB04 Unbound Unbound Unbound Unbound Unbound Unbound
1or0B02 Unbound Unbound Unbound Unbound Unbound Unbound
1or0D02 Unbound Unbound Unbound Unbound Unbound Unbound
1gk0B02 Unbound Unbound Unbound Unbound Unbound Unbound
1gk0D02 Unbound Unbound Unbound Unbound Unbound Unbound
1gk1B02 Unbound Unbound Unbound Unbound Unbound Unbound
1gk1D02 Unbound Unbound Unbound Unbound Unbound Unbound
1ghdB02 Unbound Unbound Unbound Unbound Unbound Unbound
2ae3B03 Unbound Unbound Unbound Unbound Unbound Unbound
2ae4B03 Unbound Unbound Unbound Unbound Unbound Unbound
2ae5B03 Unbound Unbound Unbound Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
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
1fm2B01 SER 170;HIS 192;ASN 413 SER 170;HIS 192;VAL 239
1jvzB01 SER 170;HIS 192;ASN 413 SER 170;HIS 192;VAL 239
1jw0B01 SER 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
1or0B01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
1or0D01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
1gk0B01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
1gk0D01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
1gk1B01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
1gk1D01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
1ghdB01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
2ae3B01 SER 1;HIS 23;ASN 244 SER 1;HIS 23;VAL 70
2ae4B01 SER 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
References Sections No. of steps in catalysis
[4]
p.1063
[10]
p.95-97

References
[1]
Resource
Comments
Medline ID
PubMed ID 11099866
Journal Biochim Biophys Acta
Year 2000
Volume 1523
Pages 123-7
Authors Lee YS, Kim HW, Lee KB, Park SS
Title Involvement of arginine and tryptophan residues in catalytic activity of glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 10991936
Journal J Biol Chem
Year 2000
Volume 275
Pages 39200-6
Authors Lee YS, Kim HW, Park SS
Title The 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.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 10945972
Journal J Struct Biol
Year 2000
Volume 131
Pages 79-81
Authors Kwon TH, Rhee S, Lee YS, Park SS, Kim KH
Title Crystallization and preliminary X-Ray diffraction analysis of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp. GK16.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 11080627
Journal Structure
Year 2000
Volume 8
Pages 1059-68
Authors Kim Y, Yoon K, Khang Y, Turley S, Hol WG
Title The 2.0 A crystal structure of cephalosporin acylase.
Related PDB 1fm2
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 11755403
Journal Chem Biol
Year 2001
Volume 8
Pages 1253-64
Authors Kim Y, Hol WG
Title Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: insight into the basis of its substrate specificity.
Related PDB 1jvz 1jw0
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 11604409
Journal J Biol Chem
Year 2001
Volume 276
Pages 48376-81
Authors Kim S, Kim Y
Title Active site residues of cephalosporin acylase are critical not only for enzymatic catalysis but also for post-translational modification.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 11782466
Journal J Biol Chem
Year 2002
Volume 277
Pages 10256-64
Authors Huang X, Zeng R, Ding X, Mao X, Ding Y, Rao Z, Xie Y, Jiang W, Zhao G
Title Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 11706000
Journal J Biol Chem
Year 2002
Volume 277
Pages 2823-9
Authors Kim Y, Kim S, Earnest TN, Hol WG
Title Precursor structure of cephalosporin acylase. Insights into autoproteolytic activation in a new N-terminal hydrolase family.
Related PDB 1keh
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID 12198140
Journal J Biol Chem
Year 2002
Volume 277
Pages 42121-7
Authors Otten LG, Sio CF, Vrielink J, Cool RH, Quax WJ
Title Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit.
Related PDB
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID 11742126
Journal Protein Sci
Year 2002
Volume 11
Pages 92-103
Authors Fritz-Wolf K, Koller KP, Lange G, Liesum A, Sauber K, Schreuder H, Aretz W, Kabsch W
Title Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C.
Related PDB 1gk1 1gk0
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 14511642
Journal Biochem Biophys Res Commun
Year 2003
Volume 310
Pages 19-27
Authors Oh B, Kim M, Yoon J, Chung K, Shin Y, Lee D, Kim Y
Title Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 14680829
Journal Biochem Biophys Res Commun
Year 2003
Volume 312
Pages 755-60
Authors Sio CF, Otten LG, Cool RH, Quax WJ
Title Analysis of a substrate specificity switch residue of cephalosporin acylase.
Related PDB
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID 12680762
Journal Biochemistry
Year 2003
Volume 42
Pages 4084-93
Authors Kim JK, Yang IS, Rhee S, Dauter Z, Lee YS, Park SS, Kim KH
Title Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation.
Related PDB 3s8r 1or0
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID 15174165
Journal Chembiochem
Year 2004
Volume 5
Pages 820-5
Authors Otten LG, Sio CF, van der Sloot AM, Cool RH, Quax WJ
Title Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase.
Related PDB
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID 15200051
Journal Protein J
Year 2004
Volume 23
Pages 197-204
Authors Mao X, Wang W, Jiang W, Zhao GP
Title His23beta and Glu455beta of the Pseudomonas sp. 130 glutaryl-7-amino cephalosporanic acid acylase are crucially important for efficient autoproteolysis and enzymatic catalysis.
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID 16446446
Journal Proc Natl Acad Sci U S A
Year 2006
Volume 103
Pages 1732-7
Authors Kim JK, Yang IS, Shin HJ, Cho KJ, Ryu EK, Kim SH, Park SS, Kim KH
Title Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries.
Related PDB 2adv 2ae3 2ae4 2ae5
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID 19800869
Journal Biochem Biophys Res Commun
Year 2009
Volume 390
Pages 342-8
Authors Cho KJ, Kim JK, Lee JH, Shin HJ, Park SS, Kim KH
Title Structural features of cephalosporin acylase reveal the basis of autocatalytic activation.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID 20606279
Journal Acta Crystallogr Sect F Struct Biol Cryst Commun
Year 2010
Volume 66
Pages 808-10
Authors Anandan A, Vallet C, Coyle T, Moustafa IM, Vrielink A
Title Crystallization and preliminary diffraction analysis of an engineered cephalosporin acylase.
Related PDB
Related UniProtKB
[19]
Resource
Comments
Medline ID
PubMed ID 21576250
Journal J Biol Chem
Year 2011
Volume 286
Pages 24476-86
Authors Yin J, Deng Z, Zhao G, Huang X
Title The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis.
Related PDB
Related UniProtKB

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.

Created Updated
2013-02-25 2016-01-27