http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
- 中文 을 입력하시려면 zhongwen을 입력하시고 space를누르시면됩니다.
- 北京 을 입력하시려면 beijing을 입력하시고 space를 누르시면 됩니다.
RISS 인기검색어
- 검색키워드
- 저자 : DasSarma, Priya (검색결과 1 건)
- 무료
- 기관 내 무료
- 유료
-
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>
연관 검색어 추천
이 검색어로 많이 본 자료
활용도 높은 자료
