Ruble James F, Lefurgy Scott T, Cornish Virginia W, Powers Rachel A
Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA.
Acta Crystallogr D Biol Crystallogr. 2012 Sep;68(Pt 9):1189-93. doi: 10.1107/S0907444912024080. Epub 2012 Aug 18.
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.
P99头孢菌素酶是一种C类β-内酰胺酶，它在一定程度上导致了细菌对β-内酰胺抗生素的广泛耐药性。该酶保守活性位点残基Asn152的突变已被证明会在体内改变β-内酰胺底物特异性。Asn152突变为甘氨酸值得注意，因为它在体内表现出底物选择性转换。为了更好地理解这种观察到的转换的结构基础，测定了P99的无辅基Asn152Gly突变体的X射线晶体结构，分辨率为1.95 Å。出乎意料的是，在活性位点观察到一个对称相关分子的人工C末端His(6)标签。His(6)标签与关键活性位点残基以及几个硫酸根离子发生了多种相互作用。此外，整个C末端占据了Asn152突变为甘氨酸后留下的空缺空间。
