| Project/Area Number |
15380074
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Saga University |
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Principal Investigator |
WATANABE Keiichi Saga University, Department of Applied Biochemistry and Food Science, Professor (40191754)
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| Co-Investigator(Kenkyū-buntansha) |
MOTOSHIMA Hiroyuki Saga University, Department of Applied Biochemistry and Food Science, Assistant Professor (20312275)
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| Project Period (FY) |
2003 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥16,100,000 (Direct Cost: ¥16,100,000)
Fiscal Year 2006: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2004: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2003: ¥8,600,000 (Direct Cost: ¥8,600,000)
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| Keywords | subtilisin / acetate kinase / cold enzyme / cold activity / psychrotroph / cold-adaptation / flexibility of enzyme / Shewanella / 結晶構造 / Pseudoalteromonas / 低温プロテアーゼ / サチライシン / X線結晶構造 / ゆらぎ / タンパク質工学 / X線結晶構造解析 / 構造ゆらぎ / ドメイン構造 / Pseudoalteromonass |
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| Research Abstract |
Enzymes produced by cold-adapted microorganisms and fish generally exhibit higher catalytic efficiency at low temperatures and lower thermostability than their mesophilic counterparts. The psychrophilic alkaline serine protease Apa1 from the Antarctic psychrotroph Shewanella sp. AS-11 consists of a subtilisin-like region (293 residues) and an additional region (148 residues) that is inserted between β8 and β9 strands of subtilisins. The crystal structure of Apa1 has been solved and refined at 1.8Å resolution. The structure of subtilisin-like domain is similar to those of subtilisins, but the insert domain has a new fold containing 11β strands and 2 α helices.
Analysis of B-factors for main chain atoms reveals a higher flexibility of the insert and the comparison of two Apa1 molecules in the asymmetric unit demonstrates a rotation of the insert relative to the subtilisin-like domain.
Comparison of Apa1 structure with the model of mesophilie homologous enzyme shows that Apa1 loses a salt b
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ridge between subtilisin-like and insert domains observed in the mesophilic counterpart. These results suggest that the flexibility of the insert contributes to the catalytic efficiency of Apa1 at low temperatures. This has been confirmed by mutational analyses of mesophilic subtilisin Carlsberg. The mutation of Pro209 to Gly at β-turn between β8 and β9 strands of subtilisin Carlsberg caused the increase in enzymatic activity.
The structural and functional properties were compared between two acetate kinases from Shewanella sp. AS-11 (SAK) and mesophilie E. coli (EAK). SAK was characterized by a shift of the optimum activity towards low temperatures and by a lower thermal stability compared with EAK. The catalytic efficiency (Kcat/Km) of SAK was 12-fold higher than that of EAK at 10℃. The activation enthalpy and entropy in both reaction directions catalyzed by SAK were lower than those by EAK, respectively. The modeled structure of SAK shows the reduced numbers of salt bridges and cation-pi interactions compared with EAK. These results indicate that SAK is cold-adapted with a more flexible structure. Less
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