DB code: S00414

RLCP classification 1.13.30015.15 : Hydrolysis
CATH domain 3.40.710.10 : Beta-lactamase Catalytic domain
E.C. 3.4.16.4
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)
3.40.710.10 : Beta-lactamase S00512 S00513 S00529 T00222

Uniprot Enzyme Name
UniprotKB Protein name Synonyms MEROPS Pfam
P15555 D-alanyl-D-alanine carboxypeptidase
DD-carboxypeptidase
DD-peptidase
EC 3.4.16.4
S12.001 (Serine)
PF00144 (Beta-lactamase)
[Graphical View]

KEGG enzyme name
serine-type D-Ala-D-Ala carboxypeptidase
DD-peptidase
D-alanyl-D-alanine-carboxypeptidase
D-alanyl-D-alanine-cleaving-peptidase
D-alanyl-D-alanine-cleaving peptidase
DD-transpeptidase
D-alanine carboxypeptidase
DD-carboxypeptidase
D-alanyl carboxypeptidase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P15555 DAC_STRSR Preferential cleavage: (Ac)(2)-L-Lys-D-Ala-|- D-Ala. Also transpeptidation of peptidyl-alanyl moieties that are N-acyl substituents of D-alanine. Secreted.

KEGG Pathways
Map code Pathways E.C.

Compound table
Substrates Products Intermediates
KEGG-id C00012 C00001 C03326 C00133 C00012 C02487 I00087 I00085 I00086
E.C.
Compound Peptide H2O (Ac)2-L-Lys-D-Ala-D-Ala D-Alanine Peptide (Ac)2-L-Lys-D-Ala Peptidyl-tetrahedral intermediate Acyl-enzyme Tetrahedral intermediate
Type peptide/protein H2O amino acids,amide group,carboxyl group,lipid,peptide/protein amino acids peptide/protein amino acids,amide group,carboxyl group,peptide/protein,lipid,peptide/protein
ChEBI 15377
270
15570
57416
269
PubChem 22247451
962
152678
71080
7311725
5462243
1cefA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1cegA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1hvbA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound Unbound Unbound Intermediate-analogue:CEH Unbound
3pteA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound 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
1cefA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 62;LYS 65;TYR 159;ASN 161 SER 62;THR 301
1cegA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 62;LYS 65;TYR 159;ASN 161 SER 62;THR 301
1hvbA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 62;LYS 65;TYR 159;ASN 161 SER 62;THR 301
3pteA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 62;LYS 65;TYR 159;ASN 161 SER 62;THR 301

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[3]
p.499
[4]
p.100-101
[5]
Scheme I, Scheme III
[6]
p.482-483
[7]
p.491-493
[8]
p.359-361
[9]
p.9534-9540
[10]
p.227-233
[11]
p.380
[12]
p.731
[13]
Scheme 3, p.1476
[15]
p.976
[16]
p.1429
[19]
p.472-476
[20]
Fig.6, p.1202-1207

References
[1]
Resource
Comments X-ray crystallography (2.8 Angstroms)
Medline ID 85207640
PubMed ID 3997832
Journal J Biol Chem
Year 1985
Volume 260
Pages 6449-58
Authors Kelly JA, Knox JR, Moews PC, Hite GJ, Bartolone JB, Zhao H, Joris B, Frere JM, Ghuysen JM
Title 2.8-A Structure of penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 and complexes with beta-lactams.
Related PDB
Related UniProtKB P15555
[2]
Resource
Comments X-ray crystallography
Medline ID 90351121
PubMed ID 2386365
Journal Antimicrob Agents Chemother
Year 1990
Volume 34
Pages 1342-7
Authors Knox JR, Pratt RF
Title Different modes of vancomycin and D-alanyl-D-alanine peptidase binding to cell wall peptide and a possible role for the vancomycin resistance protein.
Related PDB
Related UniProtKB P15555
[3]
Resource
Comments
Medline ID
PubMed ID 1546964
Journal Biochem J
Year 1992
Volume 282
Pages 495-500
Authors Hadonou AM, Jamin M, Adam M, Joris B, Dusart J, Ghuysen JM, Frere JM
Title Importance of the His-298 residue in the catalytic mechanism of the Streptomyces R61 extracellular DD-peptidase.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 1628665
Journal Eur J Biochem
Year 1992
Volume 207
Pages 97-102
Authors Hadonou AM, Wilkin JM, Varetto L, Joris B, Lamotte-Brasseur J, Klein D, Duez C, Ghuysen JM, Frere JM
Title Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 8343517
Journal Biochemistry
Year 1993
Volume 32
Pages 7278-85
Authors Jamin M, Wilkin JM, Frere JM
Title A new kinetic mechanism for the concomitant hydrolysis and transfer reactions catalyzed by bacterial DD-peptidases.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 8042992
Journal Biochem J
Year 1994
Volume 301
Pages 477-83
Authors Wilkin JM, Dubus A, Joris B, Frere JM
Title The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 8042993
Journal Biochem J
Year 1994
Volume 301
Pages 485-94
Authors Dubus A, Wilkin JM, Raquet X, Normark S, Frere JM
Title Catalytic mechanism of active-site serine beta-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 7980393
Journal Biochem J
Year 1994
Volume 303
Pages 357-62
Authors van der Linden MP, de Haan L, Dideberg O, Keck W
Title Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli.
Related PDB
Related UniProtKB
[9]
Resource
Comments X-ray crystallography (1.8/2.0 Angstroms)
Medline ID
PubMed ID 7626623
Journal Biochemistry
Year 1995
Volume 34
Pages 9532-40
Authors Kuzin AP, Liu H, Kelly JA, Knox JR
Title Binding of cephalothin and cefotaxime to D-ala-D-ala-peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum beta-lactamases.
Related PDB 1cef 1ceg
Related UniProtKB
[10]
Resource
Comments X-ray crystallography (1.6 Angstroms)
Medline ID 96083824
PubMed ID 7490745
Journal J Mol Biol
Year 1995
Volume 254
Pages 223-36
Authors Kelly JA, Kuzin AP
Title The refined crystallographic structure of a DD-peptidase penicillin-target enzyme at 1.6 A resolution.
Related PDB 3pte
Related UniProtKB P15555
[11]
Resource
Comments
Medline ID
PubMed ID 9359404
Journal Biochem J
Year 1997
Volume 327
Pages 377-81
Authors Zhao GH, Duez C, Lepage S, Forceille C, Rhazi N, Klein D, Ghuysen JM, Frere JM
Title Site-directed mutagenesis of the Actinomadura R39 DD-peptidase.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 9711239
Journal Cell Mol Life Sci
Year 1998
Volume 54
Pages 726-32
Authors Wilkin JM, Lamotte-Brasseur J, Frere JM
Title The catalytic mechanism of DD-peptidases: unexpected importance of tyrosine 280 in the transpeptidation reaction catalysed by the Streptomyces R61 DD-peptidase.
Related PDB
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID 9931012
Journal Biochemistry
Year 1999
Volume 38
Pages 1469-77
Authors Adediran SA, Pratt RF
Title Beta-secondary and solvent deuterium kinetic isotope effects on catalysis by the Streptomyces R61 DD-peptidase: comparisons with a structurally similar class C beta-lactamase.
Related PDB
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID 10393100
Journal Biochem J
Year 1999
Volume 341
Pages 409-13
Authors Rhazi N, Galleni M, Page MI, Frere JM
Title Peptidase activity of beta-lactamases.
Related PDB
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID 10986464
Journal Structure Fold Des
Year 2000
Volume 8
Pages 971-80
Authors Bompard-Gilles C, Remaut H, Villeret V, Prange T, Fanuel L, Delmarcelle M, Joris B, Frere J, Van Beeumen J
Title Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the 'penicillin-recognizing enzyme' family.
Related PDB
Related UniProtKB
[16]
Resource
Comments X-ray crystallography (1.2 Angstroms)
Medline ID
PubMed ID 11171967
Journal Proc Natl Acad Sci U S A
Year 2001
Volume 98
Pages 1427-31
Authors Lee W, McDonough MA, Kotra L, Li ZH, Silvaggi NR, Takeda Y, Kelly JA, Mobashery S
Title A 1.2-A snapshot of the final step of bacterial cell wall biosynthesis.
Related PDB 1hvb
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID 11325933
Journal J Bacteriol
Year 2001
Volume 183
Pages 3055-64
Authors Nelson DE, Young KD
Title Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID 11535797
Journal Microbiology
Year 2001
Volume 147
Pages 2571-8
Authors Reynolds PE, Ambur OH, Casadewall B, Courvalin P
Title The VanY(D) DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein.
Related PDB
Related UniProtKB
[19]
Resource
Comments
Medline ID
PubMed ID 11847270
Journal Protein Sci
Year 2002
Volume 11
Pages 467-78
Authors Wagner UG, Petersen EI, Schwab H, Kratky C
Title EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity.
Related PDB
Related UniProtKB
[20]
Resource
Comments
Medline ID
PubMed ID 12564922
Journal Biochemistry
Year 2003
Volume 42
Pages 1199-208
Authors Silvaggi NR, Anderson JW, Brinsmade SR, Pratt RF, Kelly JA
Title The crystal structure of phosphonate-inhibited D-Ala-D-Ala peptidase reveals an analogue of a tetrahedral transition state.
Related PDB 1mpl
Related UniProtKB
[21]
Resource
Comments
Medline ID
PubMed ID 12646690
Journal Protein Eng
Year 2003
Volume 16
Pages 27-35
Authors Peimbert M, Segovia L
Title Evolutionary engineering of a beta-Lactamase activity on a D-Ala D-Ala transpeptidase fold.
Related PDB
Related UniProtKB

Comments
(3) In the transition state, the tetrahedral geometry of the acyl group can be stablized by an oxyanion hole, made up by the main chain amides of Ser62 and Thr301.
This enzyme belongs to peptidase family-S12. This enzyme can act as a bifunctional enzyme, catalyzing both hydrolysis and acyl group transfer reactions (see [5]).
The catalysis involves two steps, acylation and deacylation, as follows:
(1) During the first step for acylation, the catalytic Ser62 (PDB; 1cef) acts as a nucleophile, which makes an attack on the carbonyl carbon atom to form an acyl-enzyme, accoriding to the literature [9], [20].
(2) Despite no clear evidence, Tyr159 has been thought to act as a general base, which can activate the catalytic Ser62, according to the paper [9]. On the other hand, another paper [20] proposed that Tyr159 is the general acid for formation of the tetrahedral intermediate, by protonating the leaving group, whilst the neutral Lys65 can act as a general base in the acylation step.
(4) The second step involves the deacylation, which is the hydrolysis or transfer of the intermediate. According to the paper [20], the phenoxide anion of Tyr159 would act as a general base, by activating a deacylating water for a nucleophile attack on the acyl-enzyme. Here, a positive charge on Lys65 would function as a modulator, by stabilzing the phenoxide anion on Tyr159, and by polarizing the ester bond for the nucleophilic attack by the water molecule [20].
###
Moreover, Asn161 and His298 might act as modulator for Lys65 and Tyr159, respectively.
Taken together, Tyr159 acts as acid-base, whereas Lys65 acts as base-modulator.

Created Updated
2002-09-27 2011-02-16