A Lysine-Targeted Affinity Label for Serine-?-Lactamase Also Covalently Modifies New Delhi Metallo-?-lactamase-1 (NDM-1).

ABSTRACT: The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-?-lactamases hamper the development of wide-spectrum ?-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-?-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-?-lactamases have an analogous active-site Lys residue used to bind ?-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-?-lactamases can also serve as a classical affinity label for New Delhi metallo-?-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 ?M) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-?-lactamases.

SUBMITTER: Thomas PW 

PROVIDER: S-EPMC6597302 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature