6OVZ

Crystal structure of the New Delhi metallo-beta-lactamase-1 adduct with a lysine-targeted affinity label

6OVZ の概要

• エントリーDOI: 10.2210/pdb6ovz/pdb
• 分子名称: Beta-lactamase, ZINC ION, CALCIUM ION, ... (7 entities in total)
• 機能のキーワード: ndm-1, affinity-label, covalent inhibitor, hydrolase
• 由来する生物種: Escherichia coli; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 50621.80

構造登録者

Monzingo, A.F.,Fast, W.,Thomas, P.W. (登録日: 2019-05-08, 公開日: 2019-06-12, 最終更新日: 2023-11-15)

主引用文献

Thomas, P.W.,Cammarata, M.,Brodbelt, J.S.,Monzingo, A.F.,Pratt, R.F.,Fast, W.
A Lysine-Targeted Affinity Label for Serine-beta-Lactamase Also Covalently Modifies New Delhi Metallo-beta-lactamase-1 (NDM-1).
Biochemistry, 58:2834-2843, 2019

PubMed Abstract: The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-β-lactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-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-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine K (140 μM) and k (0.045 min) 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-β-lactamases.

PubMed: 31145588
DOI: 10.1021/acs.biochem.9b00393

実験手法

X-RAY DIFFRACTION (2.017 Å)