| Project/Area Number |
17510171
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
MORIKAWA Masaaki Hokkaido Univ., Faculty of Environmental Earth Science, Prof., 大学院地球環境科学研究院, 教授 (20230104)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2005: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | Lipopeptide biosurfactant / Arthrofactin / Non-ribosomal peptide synthetase / Thioesterase domain / Gene disruption / Condensation domain / Phylogenetic tree / 非リボソーム型ペプチド合成機構 / トランスポゾン変異 / シグナル因子 / トランスポーター |
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| Research Abstract |
1.Macrocyclization in a peptide and a lipopeptide is occurred at the last step of synthesis and is usually catalyzed by a single C-terminal thioesterase (Te) domain. Arthrofactin synthetase (Arf) from Pseudomonas sp. MIS38 represents a new type of nonribosomal peptide synthetase, containing a unique tandem C-terminal Te domains with dual condensation/epimerization domain. In order to analyze the function of ArfC_Te domains in vivo, site directed mutagenesis was introduced to putative active site residues in ArfC_Te1 and ArfC_Te2. It was found that both Te domains were functional. Peaks corresponding to arthrofactin and its derivatives were completely missing in ArfC_Tel : S89A, ArfC_Tel : S89T, and ArfC_Tel : E26G/F27A mutants. Production of arthrofactin by ArfC_Te2 : S92A, ArfC_Te2 : S92A/D118A, and ArfCΔTe2 was reduced by 95 % without alteration of peptide or β-hydroxy fatty acid structure. These results suggest that Ser89 in ArfC_Tel is essential for the completion of macrocyclizati
… More
on and the release of product cyclic lipopeptide. Glu26 and Phe27 residues are also the member of active site residues in ArfC_Te1.
2.Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. A phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are L-peptidyl donors, D-peptidyl donors, and N-acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from L to D-form of incorporating amino acid acceptor occurs during or after peptide bond formation. L-peptidyl donors and D-peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process. Less
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