A point mutation leads to altered product specificity in beta-lactamase catalysis.
beta-Lactamases are the primary cause of beta-lactam antibiotic resistance in many pathogenic organisms. The beta-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme. Examination of the structure of the class A beta-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule. When N170L beta-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product. The oxazolinone subsequently undergoes rapid breakdown leading to the formation of N-phenylacetylglycine and N-formylpenicillamine. This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of beta-lactamase into that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicillin.
Lewis
E R
ER
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
Winterberg
K M
KM
Fink
A L
AL
D001216:Asparagine / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D007930:Leucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D010400:Penicillin G / Q000378:metabolism; D017354:Point Mutation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
D001216:Asparagine; D002268:Carboxypeptidases; D001618:beta-Lactamases; D007930:Leucine; D010400:Penicillin G