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- 저자 : Ram Karan (검색결과 2 건)
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Ram Karan,Raj Kumar Mohan Singh,Sanjay Kapoor,S. K. Khare 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.2
Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.7-kDa polypeptide. The deduced EMB2 protein contained a 35.4-kDa mature protein of 311 residues, with a high proportion of acidic amino acid residues. Phylogenetic analysis placed the EMB2 gene close to a known serine protease from Bacillus clausii KSM-K16. Primary sequence analysis indicated a hydrophobic inclination of the protein; and the 3D structure modeling elucidated a relatively higher percentage of small (glycine, alanine, and valine) and borderline (serine and threonine) hydrophobic residues on its surface. The structure analysis also highlighted enrichment of acidic residues at the cost of basic residues. The study indicated that solvent and salt stabilities in Geomicrobium sp. protease may be accorded to different structural features; that is, the presence of a number of small hydrophobic amino acid residues on the surface and a higher content of acidic amino acid residues, respectively.
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Laye, Victoria J.,Karan, Ram,Kim, Jong-Myoung,Pecher, Wolf T.,DasSarma, Priya,DasSarma, Shiladitya National Academy of Sciences 2017 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.114 No.47
<P><B>Significance</B></P><P>Combining comparative genomics, mutagenesis, kinetic analysis, and molecular modeling provides a powerful way to explore and understand the structure and function of proteins under extreme and potentially astrobiological conditions. Alignment of closely related cold-active and mesophilic β-galactosidase enzymes from halophilic Archaea, followed by mutagenesis and kinetic analysis, demonstrates the importance of specific amino acid residues in temperature-dependent catalytic activity, while molecular modeling provides a structural framework for their mechanism of action. Such an interdisciplinary approach shows how a very small fraction of conserved residues that are divergent from mesophilic homologs are key to enhancing catalytic activity at cold temperatures and underscores the power of combining genomics and genetics with biochemistry and structural biology for understanding polyextremophilic enzyme function.</P><P>The Antarctic microorganism <I>Halorubrum lacusprofundi</I> harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline 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 <I>csp</I>D2 promoter in the genetically tractable Haloarchaeon, <I>Halobacterium</I> sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate <I>o</I>-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 (<I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB>) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both <I>K</I><SUB>m</SUB> and <I>k</I><SUB>cat</SUB> (three substitutions) or either <I>K</I><SUB>m</SUB> or <I>k</I><SUB>cat</SUB> (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.</P>
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