Engineering a novel beta-lactamase by a single point mutation.
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.
Jacob
F
F
Laboratoire d'Enzymologie, Institut de Chimie, Université de Liège, Belgium.
Joris
B
B
Dideberg
O
O
Dusart
J
J
Ghuysen
J M
JM
Frère
J M
JM
D000215:Acylation; D001665:Binding Sites; D004795:Enzyme Stability; D007700:Kinetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010406:Penicillins / Q000378:metabolism; D015202:Protein Engineering; D012694:Serine / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D010406:Penicillins; D012694:Serine; D001618:beta-Lactamases