1999 Fiscal Year Final Research Report Summary
THE PHYSIOLOGICAL FUNCTION OF BACTERIAL LUCIFERASE AND BIOSYNTHESIS OF METHIONINE.
| Project/Area Number |
10680619
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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 |
Functional biochemistry
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| Research Institution | OSAKA CITY UNIVERSITY |
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Principal Investigator |
KASAI Sabu FACULTY OF ENGINEERING ASSOCIATE PROF., 工学部, 助教授 (90047340)
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| Project Period (FY) |
1998 – 1999
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| Keywords | lux Operon / P-flavin Binding Protein / Vibrio fischeri / Cobalamin-Dependent Methionine Synthase / FPィイD2390ィエD2 / Flavodoxin / Oxidative Stress / Ferric Uptake Regulator |
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| Research Abstract |
Occurrence of P-flavin binding protein in Vibrio fischeri and properties of the protein. We isolated P-flavin binding protein from V. fischeri. The protein was a modified luciferase, in which about 25 amino acid residues at C-terminus of the α-subunit were deleted and the prosthetic group was the fully reduced P-flavin. These results support that the physiological function of the lux operon is to produce a substitute for flavodoxin at relatively high NaCl concentrations.
The 3D structure of P-flavin blinding protein. We purified a large amount of P-flavin binding protein from V. fischeri cell extracts to prepare crystals suitable for the X-ray crystallography. This work is now in progress.
Identification of two flavodoxin genes (fldA and fldB) and ferric uptake regulator gene (fur) in Vibrio fischeri. To show the presence of flavodoxin l in V. fischeri, we identified the fldA gene in this bacteria. To determine the nucleotide sequence of the fldA, we amplified a partial fldA by PCR and t
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hen sequenced the genomic DNA upstream and downstream from the amplified region. We could complete the sequencing quite quickly using this method, which we designated as SUGDAT (Sequencing Using Genomic DNA As a Template). We also identified the fur gene downstream from the fldA gene. The fldA-fur arrangement is conserved in facultatively anaerobic bacteria classified in Proteobacteria γ-subclass and seems to be necessary for multiple control of expression of these genes.
Identification and sequence analysis of cobalamin-dependent methionine synthase (CDMS) gene (metH) in Vibrio fischeri. We proposed the presence of CDMS in luminous bacteria in the previous studies on the physiological function of the lux operon and confirmed it in this study. Two partial metH genes were amplified in both termini of the metH gene by PCR and the genomic DNA was sequenced upstream or downstream from the respective amplified region by SUGDAT to design a set of primers for PCR. A 4155 bp DNA fragment, which contains the whole metH gene, was amplified and sequenced. The amino acid sequence of the MetH protein of V. fischeri showed 74% identity with that of E. coli and all amino acid residues for cobalamin-binding found in several other organisms were conserved. Less
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Research Products
(4 results)
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[Publications] Kasai, Subu: "Identification of P-flavin Binding Protein, and the Genes for Cobalamin-Dependent Methionine Synthase and Flavodoxin l in Vibrio fischeri."Flavins and Flavoproteins 1999 (Ghisla, S., Kroneck, P. M. H., Macheroux, P., & Sund, H. Eds.) Agency for Scientific Pulications, Berlin. 333-336 (1999)
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