title: Structural analysis of the Asn152Gly mutant of P99 cephalosporinase.
abstract: P99 cephalosporinase is a class C β-lactamase that is responsible in part for the widespread bacterial resistance to β-lactam antibiotics. Mutations of the conserved active-site residue Asn152 of the enzyme have been shown to alter β-lactam substrate specificity in vivo. Mutation of Asn152 to a glycine is notable in that it exhibits in vivo substrate-selectivity switching. In order to better understand the structural basis for this observed switch, the X-ray crystal structure of the apo Asn152Gly mutant of P99 was determined to 1.95 Å resolution. Unexpectedly, the artificial C-terminal His(6) tag of a symmetrically-related molecule was observed bound in the active site. The His(6) tag makes several interactions with key active-site residues, as well as with several sulfate ions. Additionally, the overall C-terminus occupies the space left vacant upon the mutation of Asn152 to glycine.
authors.affiliation: Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Ruble
authors.forename: Scott T
authors.initials: ST
authors.lastname: Lefurgy
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Powers
mesh_terms: D002510:Cephalosporinase / Q000737:chemistry* / Q000235:genetics; D016972:Enterobacter cloacae / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein
chemical_list: D002510:Cephalosporinase