Journal Article

A DNA aptamer reveals an allosteric site for inhibition in metallo-β-lactamases

Wylie, Benjamin J., Latham, Michael P., Bui, Anthony A., Khan, Nazmul H., Xiao, Yang, Sutton, R. Bryan, Shaw, Robert W.

2019

Overview

The hydrolysis of β-lactam antibiotics by β-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-β-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-β-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-β-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-β-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 Å resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 Å from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Allosteric properties / Allosteric Site / Amides / Anti-Bacterial Agents - pharmacology / Antibiotic resistance / Antibiotics / Antimicrobial agents / Aptamers / Aptamers, Nucleotide - chemistry / Bacillus cereus / Bacillus cereus - drug effects / Bacillus cereus - enzymology / BASIC BIOLOGICAL SCIENCES / beta-Lactamase Inhibitors - pharmacology / beta-Lactamases - genetics / beta-Lactamases - metabolism / Biochemistry / Catalysis / Catalytic activity / Catalytic Domain / Cefuroxime / Chemical equilibrium / Chemistry / Crystal structure / Crystallography, X-Ray / Deoxyribonucleic acid / DNA / Docks / Drug delivery systems / Enzymatic activity / enzyme inhibitors / enzyme structure / Enzymes / Glutamine / Haddock / Hydrolysis / Inhibition / Kinetics / Lysine / Magnetic Resonance Spectroscopy / Metallo-β-lactamase / Metallography / Molecular docking / Molecular Docking Simulation / Mutagenesis, Site-Directed / Mutants / Mutation / NMR / Nuclear magnetic resonance / Numbering schemes / Organic chemistry / Pharmaceutical sciences / Protein Binding / Residues / Substrate inhibition / Substrate Specificity / β-Lactam antibiotics