1991 Fiscal Year Final Research Report Summary
Structure and function of hexulose phosphate synthase
| Project/Area Number |
02660120
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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 |
応用微生物学・発酵学
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| Research Institution | Tottori University |
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Principal Investigator |
KATO Nobuo Tottori University ; Faculty of Engineering ; Professor, 工学部, 教授 (50026556)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Hexulose phosphate synthase / Phosphohexuloisomerase / Methylotroph / ^<13>C-labeled compounds / Fructose-6-phospahte / Formaldehyde |
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| Research Abstract |
Hexulose phosphate synthase catalyzes aldol condensation of formaldehyde and ribulose 5-phosphate, which is a key enzyme in the fixation of C_1 compounds in methylotrophic bacteria. And it is suggested to be correlated to the CO_2-fixing enzyme, ribulose bisphospahte caboxylase, from the view point of the biological evolution.
Base on the amino acid sequences of the N-terminal region, three DNA probes were synthesized and used to detect the hexulose phosphate synthase gene. 5-kb EcoRl and 4-kb PstI fragments of Methylomonas aminofaciens 77a that is a facultative methylotroph was detected in Southern hybridization with the probes. Three fragments were cloned into pUC118 by colony hybridization. Further Southern hybridization revealed that the fragments contained the hexulose phosphate synthase gene. The sequencing of the positive clones are in proceeding.
The formaldehyde-fixing system of a methonol-using bacterium, Methylomonas aminofaciens 77a was used for the production of ^<13>C-labeled D-fructose-6-phosphate from ^<13>C-formaldehyde and D-ribose-5-phosphate. The specific and high yield production of[1- ^<13>C]D-fructose-6-phosphate was performed using an enzyme system that contained partially purified hexulose phosphate synthase and phosphohexuloisomerase from the bacterium, and spinach phosphoriboisomerase. A fed-batch reaction, in which 50 mM formaldehyde and 75 mM D-ribose-5-phosphate were added at 15-min intervals, afforded 203 mM D-fructose-6-phosphate for 90 min.
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