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Engineering a novel beta-lactamase by a single point mutation.

F Jacob·B Joris·O Dideberg·J Dusart·J M Ghuysen·J M Frère
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Abstract

beta-Lactamases are widespread and efficient bacterial enzymes which play a major role in bacterial resistance to penicillins and cephalosporins. In order to elucidate the role of the residues lying in a conserved loop of the enzymatic cavity of the active-site serine Streptomyces albus G beta-lactamase, modified proteins were produced by oligo-directed mutagenesis. Mutation of Asn116, which lies on one side of the active site cavity pointing to the substrate-binding site, into a serine residue resulted in spectacular modifications of the specificity profile of the enzyme. That replacement yielded an enzyme with a nearly unchanged activity towards good penicillin substrates. In sharp contrast its efficiency in hydrolysing cephalosporins was drastically reduced, the best substrates suffering the largest decrease in the second-order rate constant for serine acylation. In fact that single mutation generated a truly new enzyme behaving exclusively as a penicillinase, a situation which is never encountered to the same degree in any of the numerous naturally occurring variants of class A beta-lactamases.

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AcylationBinding SitesEnzyme StabilityKineticsMolecular StructureMutagenesis, Site-DirectedPenicillinsProtein EngineeringSerineStreptomycesSubstrate Specificitybeta-LactamasesJournal ArticleResearch Support, Non-U.S. Gov't

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F Jacob, B Joris, O Dideberg, J Dusart, J M Ghuysen, J M Frère. 1990. Engineering a novel beta-lactamase by a single point mutation.. https://doi.org/10.1093/protein%2F4.1.79

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