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Protein Science (2007), 16:2636-2646. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Saturation mutagenesis of Asn152 reveals a substrate selectivity switch in P99 cephalosporinase

Scott T. Lefurgy1,3, René M. de Jong2, and Virginia W. Cornish2

1 Department of Biological Sciences, Columbia University, New York, New York 10027, USA
2 Department of Chemistry, Columbia University, New York, New York 10027, USA

(RECEIVED June 27, 2007; FINAL REVISION September 14, 2007; ACCEPTED September 14, 2007)

In class C beta-lactamases, the strictly conserved Asn152 forms part of an extended active-site hydrogen-bonding network. To probe its role in catalysis, all 19 mutants of Enterobacter cloacae P99 cephalosporinase Asn152 were simultaneously constructed and screened in Escherichia coli for their in vivo activity. The screen identified the previously uncharacterized mutants Asn152Ser, Asn152Thr, and Asn152Gly, which possess significant activity and altered substrate selectivity. In vitro measurement of Michaelis-Menten kinetic constants revealed that the Asn152Ser mutation causes a selectivity switch for penicillin G versus cefoxitin. Asn152Thr showed a 63-fold increase in k cat for oxacillin, a slow substrate for wild-type cephalosporinase. The results contribute to a growing body of data showing that mutation of highly conserved residues in the active site can result in substrate selectivity changes. The library screening method presented here would be applicable to substrate selectivity determination in other readily screenable enzymes.

Keywords: beta-lactamase; substrate selectivity; microbial sensitivity tests; saturation mutagenesis; kinetics; enzymology


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