title: Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15.
abstract: A variety of CTX-M-type extended-spectrum β-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of β-lactam resistance in the host strains. All tested enzymes were susceptible to serine β-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A(77)V, displayed enhanced catalytic activity (kcat/Km) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A(77)V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.
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 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: 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: 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: Lanping
authors.initials: L
authors.lastname: Liu
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: Xiaojie
authors.initials: X
authors.lastname: Chen
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: Li
authors.initials: L
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@inet.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@inet.polyu.edu.hk.
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D020134:Catalytic Domain; D002645:Chickens; D057075:Enzyme Assays; D004926:Escherichia coli / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D047090:beta-Lactams; C456080:beta-lactamase CTX-M-14; C473109:beta-lactamase CTX-M-15; D001618:beta-Lactamases