1995 Fiscal Year Final Research Report Summary
Protein engineering for the change of substrate specificity of the ethylene forming enzyme of Pseudomonas syringae
| Project/Area Number |
06650928
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物・生体工学
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| Research Institution | Kumamoto Institute of Technology |
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Principal Investigator |
OGAWA Takahira KUMAMOTO INSTITUTE OF TECHNOLOGY,Dep.Applied Microbial Technology, Professor, 工学部, 教授 (40029244)
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| Co-Investigator(Kenkyū-buntansha) |
NAGAHAMA Kazuhiro KUMAMOTO INSTITUTE OF TECHNOLOGY,Dep.Applied Microbial Technology, Assistant, 工学部, 助手 (50248605)
MASTUOKA Masayoshi KUMAMOTO INSTITUTE OF TECHNOLOGY,Dep.Applied Microbial Technology, Associate Pro, 工学部, 助教授 (10121667)
FUKUDA Hideo KUMAMOTO INSTITUTE OF TECHNOLOGY,Dep.Applied Microbial Technology, Professor, 工学部, 教授 (10150830)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Ethylene / Ethylene-forming en2yme / Pseudornonas syringal / Histidine / Protein engineering |
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| Research Abstract |
1. The roles of ten histidine residues in the ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 were studied individually by site-directed mutagenesis. All ten histidine residues in the EFE were sequentially replaced by glutamine residues. Only mutations at H-189 and H-233 caused a total loss of activity, while, mutations at H-268 and H-284 resulted in enzymes with 1.3% and 2.6% of the control activity in vitro. Taken together with the kinetic studies and inactivation studies, the results indicate that H-189 and H-233 might play the most important roles, being involved, in the binding of iron, while H-268 might play an important role in binding of substrate.
2. Hydropathy plot of P.syringae EFE and plant type EFE shows that both look like similar when hydrophobic region (Pro93-Pro114) and hydrophilic region (Arg209-Ser226) are removed from P.syringae EFE.Therefore, two regions of P.syringae were tried to remove from P.syringae EFE as shown below.
(1) Preparation of two omitted EFE genes by PCR method.
(2) Ligation to pUC18 with an omitted EFE gene, respectively.
(3) Transformation of Escherichia coli using an omitted EFE gene.
Ethylene-forming activities were measured. But, at present, we could not observe EFE activity against 2-oxoglutarate and/or ACC.
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