The role of OXA-1 beta-lactamase Asp(66) in the stabilization of the active-site carbamate group and in substrate turnover.
The OXA-1 beta-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser(67). In class A beta-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp(66) in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp(66) mutants expressed in Escherichia coli DH10B ranged from < or =8 microg/ml for cysteine, proline and the basic amino acids to > or =256 microg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys(70), that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria-Bertani-broth agar resulted in a > or =16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp(66) forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys(70) and thereby profoundly affects substrate turnover.
Leonard
David A
DA
Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA.
Hujer
Andrea M
AM
Smith
Brian A
BA
Schneider
Kyle D
KD
Bethel
Christopher R
CR
Hujer
Kristine M
KM
Bonomo
Robert A
RA
D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002219:Carbamates / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D002219:Carbamates; D017931:DNA Primers; D001224:Aspartic Acid; C039404:beta-lactamase OXA-2; D001618:beta-Lactamases