Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the 'penicillin-recognizing enzyme' family.
BACKGROUND
beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis. The most common bacterial resistance mechanism against beta-lactam compounds is the synthesis of beta-lactamases that hydrolyse beta-lactam rings. These enzymes are believed to have evolved from cell-wall DD-peptidases. Understanding the biochemical and mechanistic features of the beta-lactam targets is crucial because of the increasing number of resistant bacteria. DAP is a D-aminopeptidase produced by Ochrobactrum anthropi. It is inhibited by various beta-lactam compounds and shares approximately 25% sequence identity with the R61 DD-carboxypeptidase and the class C beta-lactamases.


RESULTS
The crystal structure of DAP has been determined to 1.9 A resolution using the multiple isomorphous replacement (MIR) method. The enzyme folds into three domains, A, B and C. Domain A, which contains conserved catalytic residues, has the classical fold of serine beta-lactamases, whereas domains B and C are both antiparallel eight-stranded beta barrels. A loop of domain C protrudes into the substrate-binding site of the enzyme.


CONCLUSIONS
Comparison of the biochemical properties and the structure of DAP with PBPs and serine beta-lactamases shows that although the catalytic site of the enzyme is very similar to that of beta-lactamases, its substrate and inhibitor specificity rests on residues of domain C. DAP is a new member of the family of penicillin-recognizing proteins (PRPs) and, at the present time, its enzymatic specificity is clearly unique.
Bompard-Gilles
C
C
Laboratorium voor Eiwitbiochemie en Eiwitengineering, Rijksuniversiteit-Gent, K.L. Ledeganckstraat, 35, B-9000, Gent, Belgium.
Remaut
H
H
Villeret
V
V
Prangé
T
T
Fanuel
L
L
Delmarcelle
M
M
Joris
B
B
Frère
J
J
Van Beeumen
J
J
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D018360:Crystallography, X-Ray; D019281:Dimerization; D006602:Hexosyltransferases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry; D020621:Ochrobactrum anthropi / Q000201:enzymology*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000201:enzymology; D001618:beta-Lactamases / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; D000626:Aminopeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases