1999 Fiscal Year Final Research Report Summary
Characterization of Biotin Transporters
| Project/Area Number |
10680569
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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 |
Bioorganic chemistry
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| Research Institution | Kyushu Institute of Technology |
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Principal Investigator |
KONDO Hiroki Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Professor, 情報工学部, 教授 (60038057)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Biotin / Vitamin / Coenzyme / Transporter / Carboxylase / Carboxylation / Genetic Engineering / Protein Engineering |
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| Research Abstract |
Biotin is a vitamin and cofactor and plays an important role in such biological processes as fatty acid biosynthesis. Biotin is either synthesized within the cells or transported across plasma membranes and then attached to a specific lysine residue of biotin-dependent enzymes. Hence, malfunctioning of either one or all of these processes as well as defects in biotin-requiring proteins result in severe disorders in organisms. In order to clarify the function of biotin in cellular processes in more detail, comprehensive studies were undertaken in this project with special emphasis on biotin transport across cell membranes. Biotin transporters have been studied extensively in Escherichia coli and yeast, Saccharomyces cerevisiae, but their chemical entities escaped identification. On the basis of previous knowledge on the E. coli transporter that its gene (bioP) resides at 86 min on the genetic map, data base was searched for a probable candidate(s). It was found that gene o461 meets all
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the requirements for a (biotin) transporter; the deduced amino acid sequence suggests that it is a hydrophobic protein and shows considerable homology with those of known amino acid transporters from various sources. Disruption of this gene resulted in a significant decrease in the biotin transport activity of E. coli. In addition to the biotin transport in E. coli, the catalytic function of biotin was studied with acetyl-CoA carboxylase (ACC) and pyruvate carboxylase (PC). E. coli ACC is made of three components: biotin carboxylase (BC), carboxyl transferase and biotin carboxyl carrier protein (BCCP), while PC from Bacillus stearothermophilus is of a single polypeptide chain. Two approaches were taken, (l) site-directed mutagenesis of putative active site residues and (2) construction of chimeric enzymes of ACC and PC. In the latter, functional domains BC and BCCP of PC were replaced with the corresponding domains of ACC. Preliminary studies of these chimeras presented information on how biotin interacts with the individual domains of the carboxylases. Less
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