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Publication Abstract from PubMed

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-beta-lactamases hamper the development of wide-spectrum beta-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-beta-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-beta-lactamases have an analogous active-site Lys residue used to bind beta-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-beta-lactamases can also serve as a classical affinity label for New Delhi metallo-beta-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 muM) 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 A 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-beta-lactamases.

A Lysine-Targeted Affinity Label for Serine-beta-Lactamase Also Covalently Modifies New Delhi Metallo-beta-lactamase-1 (NDM-1).,Thomas PW, Cammarata M, Brodbelt JS, Monzingo AF, Pratt RF, Fast W Biochemistry. 2019 Jun 7. doi: 10.1021/acs.biochem.9b00393. PMID:31145588^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.