Analysis of the thermostable hydrogenase from Hydrogenovibrio marinus and an attempt for hydrogenase stabilization
| Project/Area Number |
13836001
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
応用微生物学
|
|---|
| Research Institution | Ibaraki University |
|---|
Principal Investigator |
NISHIMURA Hirofumi IBARAKI UNIVERSITY, College of Agriculture, Associate Professor, 農学部, 助教授 (10260465)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KURUSU Yasurou IVARAKI UNIVERSITY, College of Agriculture, Professor, 農学部, 教授 (60272118)
|
|---|
| Project Period (FY) |
2001 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
|
|---|
| Keywords | thermal stability / hydrogenase / hydrogen-oxidizing bacterium / site-specific mutagenesis / stabilization / 安定化 / 部位特異的変見 |
|---|
| Research Abstract |
The deduced protein sequence of the membrane-bound hydrogenase (MBH) from Hydrogenovibrio marinus revealed high homology with other [NiFe]-hydrogenases. All conserved motifs and typical signatures of standard [NiFe]-hydrogenases were found. However, increase of Ile-residues especially in the catalytic subunit HoxG was observed as a feature of H.Marinus MBH. Most of such residues were assigned to be located inside the protein or at subunit interface in the homology model of H.marinus MBH that was constructed according to the crystal structure of Desulfovibrio vulgaris hydrogenase. Amino acid replacement with Ile residue in the relevant positions in R.eutropha HoxG resulted in enhancement of thermal stability. P181I or L243I substitution in R.eutropha HoxG, locating at subunit interface, significantly increased thermal stability of methyleneblue reducing activity, suggesting stabilization of subunit association presumably due to increase of hydrophobic interaction. These mutations, as well as mutations in the hydrophobic core of HoxG ([V115I], [V253I] and [N168I, L170I]), were also effective for thermal stabilization of the active center. Cavity-packing effect for protein stabilization was additionally expected for several mutant MBHs. Ile-residues in the H.marinus HoxG focused for amino acid substitutions construct so-called "Ile-clusters" with several Ile residues locating near-by, which must form a stable hydrophobic core structure. By the analysis of the downstream region of H.Marinus MBH structural gene, ORFs coding for cytochrome b-like protein and a novel membrane protein that was speculated as an electron carrier related to H_2-oxidation were found. Two ORFs coding for proteins that are speculated to be necessary for MBH maturation were also found. In this study, thermal stability of hydrogenase was successfully improved without loss in catalytic activity, which suggested an useful strategy for hydrogenase stabilization.
|
Report
(4 results)
Research Products
(7 results)
