2002 Fiscal Year Final Research Report Summary
Analyses of the crystal structure of 4-α-glucanotransferase and its production mechanism of new cycloamylose
| Project/Area Number |
12460047
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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 |
応用微生物学・応用生物化学
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| Research Institution | Aomori University |
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Principal Investigator |
MATSUZAWA Hiroshi Aomori University, Department of Bioscience and Biotechnology, Professor, 工学部, 教授 (00011966)
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| Co-Investigator(Kenkyū-buntansha) |
FUSHINOBE Shinya The University of Tokyo, Department of Biotechnology, Assistant Professor, 大学院・農学生命科学研究科, 助手 (00302589)
WAKAGI Takayoshi The University of Tokyo, Department of Biotechnology, Associate Professor, 大学院・農学生命科学研究科, 助教授 (70175058)
KAMIIE Katsuyoshi Aomori University, Department of Bioscience and Biotechnology, Assistant Professor, 工学部, 助手 (70275519)
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| Project Period (FY) |
2000 – 2002
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| Keywords | 4-α-Glucanotransferase / Cycloamylose / X-ray crystal analysis / Crystal structure / Glycoside hydrolase family / Acarbose / Substrate-enzyme complex structure / Archaeon |
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| Research Abstract |
4-α-Glucanotransferase of Thermococcus litoralis, a hyperthermophilic archeon, consists of 659 amino acid residues. The enzyme catalyzes not only intermolecular transglycosylation (so-called disproportionation) to produce linearα-1,4-glucans with various length of glucose units, but also intramolecular transglycosylation to produce cyclicα-1,4-glucans (cycloamyloses) with 16 to several hundred glucose units from linear amylose. Following results were obtained in this research.
(1) The enzyme was composed of two domains; an N-terminal domain (residues 1-381), which contained a (β/α)_7 barrel fold, and a C-terminal domain (residues 389-659), which had a twistedβ-sandwich fold.
(2) Glu123 and Asp214 were found to be the catalytic nucleophile and acid/base catalyst, respectively, through biochemical and crystal structure analyses. The catalytic residues were located in the cleft of the N-terminal domain, indicating that the N-terminal domain is a catalytic domain of the enzyme.
(3) On the structure of a complex between the enzyme and acarbose (an enzyme inhibitor), the acarbose molecule bound to subsites -1 to +3. A maltose molecule was also found to bind at the edge of the active site cleft; the binding site corresponds to subsites -5 and -6. The enzyme was revealed to possess at least nine subsites, -6 to +3.
(4) The active site cleft of the enzyme was tunnel-like in shape, as evidenced by the three lids that covered the cleft. The first lid (residues 220-224) protruded from the (β/α)^7 barrel. The second (residues 358-363) and third (627-630) lids protruded from a three-helix bundle and the C-terminal domain, respectively. Upon binding of acarbose, the conformation of lids 2 and 3 changed significantly.
(5) It seemed that the enzyme produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft.
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