| Project/Area Number |
16380079
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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 |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
SAKATA Kanzo Kyoto University, Institute for Chemical Research, Professor (20087563)
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| Co-Investigator(Kenkyū-buntansha) |
HIRATAKE Jun KYOTO UNIVERSITY, Institute for Chemical Research, Associate professor (80199075)
MIZUTANI Masaharu KYOTO UNIVERSITY, Inst. for Chemical Research, Assistant professor (60303898)
SHIMIZU Bunichi KYOTO UNIVERSITY, Inst. for Chemical Research, Assistant professor (50324695)
KATO Hiroaki KYOTO UNIVERSITY, Graduate School of Pharmacuitical Sciences, Professor (90204487)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2006: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2005: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2004: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Keywords | primeverosidase / furcatin hydrolase / vicianin hydrolase / Family 1 glycosyl hydrolase / X-ray crystallography / phylogenetic analysis / diglycosidase / molecular evolution / β-プリメベロシダーゼ / ファミリー1 グリコシダーゼ / β-プリメベロシルアミジン / ファミリー1 / アフィニティー吸着体 / βプリメベロシダーゼ / カラスノエンドウ / ムシカリ / グリコシルアミジン / チャ葉 |
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| Research Abstract |
1) Clarification of substrate specificities and catalytic mechanisms of diglycosidases
1-a) X-Ray crystallographic analysis of β-primeverosidase from a tea plant
We have succeeded in synteshis of a potent glycosidase inhibitor, β-primeverosyl amidine, for a representative diglycosidase, β-primeverosidase. Single crystalls of β-primeverosidase binding the inhibitor was obtained and subjected to X-ray crystallographic analysis to obtain the structure of the protein with resolution of 1.8 A based on the structure of a family 1 β-glucosidase from maize. The structure allowed us to identify the amino acid residures that interact with disaccharide moiety of the substrates and to show the detailed structure of the opening part of catalytic site. Based on these observations and the substrate specificities of the enzyme we could have understood the substrate recognition mechanism of the enzyme in detail.
1-b) Clarification of the substrate recognition mechanism of a diglycosidase by the use of 6'-
… More
substituted β-D-glucopyranosides
Pseudo-disaccharide glycosides with various kinds of S-substituents (alcohols of C_<2_4> and D-xylopyranosyl) at C-6'of p-nitrophenyl (pNP) β-D-glucopyranosides were synthesized and subjected to hydrolysis by a diglycosidase, vicianin hydrolase. This diglycosidase shows high reactivity against pNP β-primeveroside, but little activity against 6'-S-substituted pNP β-primeveroside as well as pNPβ-D-glucopyranoside. This enzyme shows high activity against pseudo-diglycosides with a straight chain C3-alcohol substituent at C-6'. These observations suggest that the hydroxy group of the C_3-alcohol substituent interacts with the amino acid residues concerned with the recognition of the disaccharide moeiety of natural substrates.
2) Distribution of diglycosidases in plant kingdom and analysis of molecular evolution of diglycosidases
By the use of pNP P-primeveroside as a substrate diglycosidases were screened among plant crude enzymes randomly selected. Out of 18 kinds of plant species β-primeverosidase-like activity was found in the crude enzyme of 7 kinds of plants, indicating considerably wide distribution of diglycosidases among plant kingdom.
Phylogenetic tree analysis of diglycosidases, β-primeverosidase, furcatin hydrolase and vicianin hydrolase in family 1 of glycosyl hydrolases were carried out based on their full amino acid sequences, indicating that these diglycosidases cluster with each other. Less
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