globalchange  > 气候变化与战略
DOI: 10.1073/pnas.1711542114
论文题名:
Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase
作者: Laye V.J.; Karan R.; Kim J.-M.; Pecher W.T.; DasSarma P.; DasSarma S.
刊名: Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
出版年: 2017
卷: 114, 期:47
起始页码: 12530
结束页码: 12535
语种: 英语
英文关键词: Enzyme kinetics ; Extremophile ; Haloarchaea ; Psychrophile ; Site-directed mutagenesis
Scopus关键词: amino acid ; beta galactosidase ; chromogenic substrate ; 2-nitrophenylgalactoside ; 4 nitrophenylgalactoside ; archaeal protein ; beta galactosidase ; protein binding ; recombinant protein ; Antarctica ; Article ; catalytic efficiency ; cold ; controlled study ; enzyme activity ; enzyme kinetics ; enzyme purification ; gene synthesis ; Halobacteriales ; Haloferax ; Haloferax lucentense ; Haloferax volcanii ; Halorubrum lacusprofundi ; Haloterrigena turkmenica ; hydrogen bond ; kinetics ; Natronococcus occultus ; nonhuman ; priority journal ; promoter region ; restriction fragment ; site directed mutagenesis ; steady state ; temperature sensitivity ; alpha helix ; amino acid substitution ; beta sheet ; chemistry ; cold ; enzyme active site ; enzyme specificity ; enzymology ; gene expression ; genetics ; Halobacterium ; Halorubrum ; metabolism ; molecular cloning ; molecular model ; protein domain ; structure activity relation ; thermodynamics ; X ray crystallography ; Amino Acid Substitution ; Antarctic Regions ; Archaeal Proteins ; beta-Galactosidase ; Catalytic Domain ; Cloning, Molecular ; Cold Temperature ; Crystallography, X-Ray ; Gene Expression ; Halobacterium ; Halorubrum ; Kinetics ; Models, Molecular ; Mutagenesis, Site-Directed ; Nitrophenylgalactosides ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Recombinant Proteins ; Structure-Activity Relationship ; Substrate Specificity ; Thermodynamics
英文摘要: The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic β-galactosidase that functions in cold, hy-persaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Hal-oarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme. © 2017, National Academy of Sciences. All rights reserved.
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资源类型: 期刊论文
标识符: http://119.78.100.158/handle/2HF3EXSE/163773
Appears in Collections:气候变化与战略

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作者单位: Laye, V.J., Institute of Marine and Environmental Technology, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21202, United States; Karan, R., Institute of Marine and Environmental Technology, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21202, United States; Kim, J.-M., Institute of Marine and Environmental Technology, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21202, United States; Pecher, W.T., Institute of Marine and Environmental Technology, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21202, United States; DasSarma, P., Institute of Marine and Environmental Technology, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21202, United States; DasSarma, S., Institute of Marine and Environmental Technology, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21202, United States

Recommended Citation:
Laye V.J.,Karan R.,Kim J.-M.,et al. Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase[J]. Proceedings of the National Academy of Sciences of the United States of America,2017-01-01,114(47)
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