title: Probing the effect of the non-active-site mutation Y229W in New Delhi metallo-β-lactamase-1 by site-directed mutagenesis, kinetic studies, and molecular dynamics simulations.
abstract: New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactamases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1 ∼ 7 fold increases in k(cat) /K(m) values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.
authors: School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China.
authors: Jiao
authors: J
authors: Chen
authors: Hui
authors: H
authors: Chen
authors: Yun
authors: Y
authors: Shi
authors: Feng
authors: F
authors: Hu
authors: Xingzhen
authors: X
authors: Lao
authors: Xiangdong
authors: X
authors: Gao
authors: Heng
authors: H
authors: Zheng
authors: Wenbing
authors: W
authors: Yao
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1