title: Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes.
abstract: Clinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between the blaCTX-M-15 and blaCTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity.
authors.affiliation: College of Veterinary Medicine, Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, National Reference Laboratory of Veterinary Drug Residues (SCAU), South China Agricultural University, Guangzhou, People's Republic of China Shenzhen Key Laboratory for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong Polytechnic University, Shen Zhen Research Institute, Shenzhen, People's Republic of China.
authors.forename: Dandan
authors.initials: D
authors.lastname: He
authors.affiliation: Shenzhen Key Laboratory for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong Polytechnic University, Shen Zhen Research Institute, Shenzhen, People's Republic of China State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR.
authors.forename: Jiachi
authors.initials: J
authors.lastname: Chiou
authors.affiliation: College of Veterinary Medicine, Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, National Reference Laboratory of Veterinary Drug Residues (SCAU), South China Agricultural University, Guangzhou, People's Republic of China.
authors.forename: Zhenling
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: Shenzhen Key Laboratory for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong Polytechnic University, Shen Zhen Research Institute, Shenzhen, People's Republic of China State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR.
authors.forename: Edward Wai-Chi
authors.initials: EW
authors.lastname: Chan
authors.affiliation: College of Veterinary Medicine, Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, National Reference Laboratory of Veterinary Drug Residues (SCAU), South China Agricultural University, Guangzhou, People's Republic of China jhliu@scau.edu.cn sheng.chen@polyu.edu.hk.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Liu
authors.affiliation: Shenzhen Key Laboratory for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong Polytechnic University, Shen Zhen Research Institute, Shenzhen, People's Republic of China State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR jhliu@scau.edu.cn sheng.chen@polyu.edu.hk.
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D007527:Isoenzymes; D011994:Recombinant Proteins; D047090:beta-Lactams; C456080:beta-lactamase CTX-M-14; D001618:beta-Lactamases