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 15377	270 270	15570 57416 15570 57416		269 269
PubChem		22247451 962 22247451 962	152678 152678	71080 7311725 71080 7311725		5462243 5462243

1cefA	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1cegA	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1hvbA	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Intermediate-analogue:CEH	Unbound
3pteA	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	SER 62;LYS 65;TYR 159;ASN 161			SER 62;THR 301
1cegA	SER 62;LYS 65;TYR 159;ASN 161			SER 62;THR 301
1hvbA	SER 62;LYS 65;TYR 159;ASN 161			SER 62;THR 301
3pteA	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.

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