title: E152A substitution drastically affects NDM-5 activity.
abstract: New Delhi Metallo beta-lactamase (NDM) is of significant public health concern due to its enormous potential to hydrolyse all major beta-lactams including carbapenems. Amino acid substitutions outside the active site reportedly affect NDM beta-lactamase activities. Here, the effect of amino acid substitutions in the possible omega-like loop region of NDM-5 has been elucidated. Overall, three substitution mutations near active site of NDM-5 were done, namely, E152A, S191A and D223A and subsequently, the change in antimicrobial resistance was monitored upon expressing each mutant in a suitable host. Among the three mutants, E152A substitution on a loop near the active site resulted in significant reduction in beta-lactam antibiotic resistance as compared to NDM-5 that compelled us to conduct further studies on the E152A-substituted NDM-5. The purified NDM-5 was able to hydrolyse all the beta-lactams tested whereas the E152A mutation suppressed its activities. NDM-5 showed maximum kcat/Km ratio against penicillins and carbapenems and had lower Km as compared to NDM-5_E152A. Though, the amino acid substitution did not affect the overall folding pattern of NDM-5, significant differences in thermal stability between the wild-type and mutated protein were observed. Therefore, we infer that the E152 residue is important in regulating the beta-lactam hydrolysing properties of NDM-5.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Kumar
authors.affiliation: UFR Biosciences, Université Félix Houphouët-Boigny, 22 BP 582 Abidjan 22, Côte d'Ivoire.
authors.forename: Bagre
authors.initials: B
authors.lastname: Issa
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
authors.forename: Debasish
authors.initials: D
authors.lastname: Kar
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
authors.forename: Sarmistha
authors.initials: S
authors.lastname: Biswal
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
authors.forename: Anindya S
authors.initials: AS
authors.lastname: Ghosh
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015780:Carbapenems / Q000494:pharmacology; D020134:Catalytic Domain; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D018698:Glutamic Acid / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D009154:Mutation; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D047090:beta-Lactams; D018698:Glutamic Acid; C000615639:beta lactamase NDM-5, E coli; D001618:beta-Lactamases
keywords: beta-lactam; carbapenemase; metallo beta-lactamases; thermal stability