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O Ifuku

Publications and source records attributed to O Ifuku.

7 recordsLinked to original sources
PubMed1999

Characterization of the promoter region of the human melanocortin-1 receptor (MC1R) gene.

We sequenced 3201 bp upstream from the ATG translation start codon of the human melanocortin-1 receptor (MC1R). A number of transcriptional initiation sites were detected over a region of approximately 600 base pairs upstream of the receptor coding region. These consist of GC-rich regions, each including SP-1 consensus binding motifs. Neither a TATA nor a CAAT box was found in this region. The 5'-flanking region also contains the consensus regulatory elements for AP-1, AP-2, and several E-boxes. Gel shift assays targeting the three GC boxes confirmed binding of SP-1. A promoter assay revealed that the minimal region exhibiting promoter activity was located between nucleotides -517 and -282 in human melanoma SK-Mel-2 cells. Further deletion from -517 to -447, which removed an SP-1 site, completely abolished luciferase activity. In conclusion, the MC1R promoter shares the characteristics of many other GPCR promoters. These characteristics include GC-rich sequence, lack of a TATA box, and binding of SP-1.

Base Sequence↗
PubMed1995

Molecular analysis of growth inhibition caused by overexpression of the biotin operon in Escherichia coli.

Constitutive overexpression of the biotin operon (type 9 mutation) in a multicopy plasmid resulted in growth inhibition in Escherichia coli. Deletion analysis of the biotin operon indicated that overexpression of the bioB gene alone, the product of which is believed to catalyze the conversion of dethiobiotin to biotin, is sufficient for growth inhibition. This growth inhibition was still observed when the wild-type bioB gene was replaced by several mutant-type bioB genes derived from biotin auxotrophs that have base-pair substitutions creating amino acid substitutions in the bioB gene product. However, the modification of Ala 143 and Gly 99 of the bioB gene product resulted in recovery from growth inhibition. These results suggest that this phenotype of growth inhibition by overexpression of the bioB gene in E. coli is independent of the biotin-forming activity itself, but is caused by some function involving a specific conformation of the bioB gene product.

Amino Acid Sequence↗
PubMed1994

Flavodoxin is required for conversion of dethiobiotin to biotin in Escherichia coli.

We have reported [Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M. & Fukushima, S. (1992) Biosci. Biotechnol. Biochem. 56, 1780-1785] the enzymic conversion of dethiobiotin to biotin (catalyzed by the enzyme encoded by bioB) in cell-free extract of Escherichia coli which had been genetically engineered for high bioB expression. An unidentified protein(s) in addition to the bioB gene product is obligatory for this reaction. We have found that this protein was precipitated from the cell-free extract with poly(ethyleneimine), and we have purified it to homogeneity by a procedure which includes ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration, and Mono Q chromatography. The apparent molecular mass of the purified protein was estimated to be about 21 kDa by SDS/PAGE. The N-terminal amino acid sequence of the purified protein was identical with that of E. coli flavodoxin. We conclude that flavodoxin is required for conversion of dethiobiotin to biotin in E. coli. Studies with purified flavodoxin and the fraction containing the bioB gene product suggested that protein(s) in addition to the bioB gene product and flavodoxin is also obligatory for the reaction.

Amino Acid Sequence↗
PubMed1994

Origin of carbon atoms of biotin. 13C-NMR studies on biotin biosynthesis in Escherichia coli.

The origin of the carbon atoms of pimeloyl-CoA, the earliest known precursor in the pathway of de novo biotin biosynthesis in Escherichia coli, was investigated by 13C-NMR spectroscopy. In fermentation of the biotin-overproducing DRK332/pXBA312 strain of Escherichia coli (a repressor mutant carrying a biotin operon fragment in the plasmid), a high dose of L-alanine (8 g/l) stimulated dethiobiotin and biotin accumulation. Although L-alanine is a known precursor of 7-keto-8-aminopelargonic acid in biotin biosynthesis, the 13C-NMR spectrum of dethiobiotin showed that the C-3 of L-[3-13C]alanine was incorporated into not only the methyl carbon (C-9) but also alternate carbons (C-2, C-4, C-6) of the side chain, and these latter positions are the same as those labeled with D-[1-13C]glucose. These data indicate that L-alanine can act as an alternative carbon source, suggesting that acetyl-CoA is a possible precursor for pimeloyl-CoA synthesis. In accordance with this hypothesis, the C-1 of sodium (1-13C)acetate and the C-2 of sodium (2-13C)acetate were incorporated into alternate carbons in the side chain of dethiobiotin, i.e., (C-1, C-3, C-5, C-7) and (C-1, C-2, C-4, C-6), respectively. These results suggested firstly that in E. coli pimeloyl-CoA is biosynthesized from L-alanine and/or acetate via acetyl-CoA, but not via pimelic acid, which has been suggested as a biotin precursor in other species, and secondly that the carboxyl group of biotin originates from carbon dioxide produced through the tricarboxylic acid cycle.

Acetates↗
PubMed1993

Sequencing analysis of mutation points in the biotin operon of biotin-overproducing Escherichia coli mutants.

We analyzed mutation points of the biotin operon from biotin-overproducing mutants of Escherichia coli resistant to two biotin analogs, actithiazic acid and 5-(2-thienyl)-valeric acid, by DNA sequencing. The biotin operons cloned from these mutants were classified into three groups. One point mutation, which was a GC-->AT change within the operator overlapping the -10 region of the rightward (bioB) promoter, was considered to result in disruption of operator structure and enhancement of promoter activity. Two other point mutations, which were both GC-->AT changes just before and after the initiation codon of the bioB gene, were considered to activate the translation efficiency. These mutations significantly accelerated the biotin-forming activity from dethiobiotin in cell-free extracts.

Base Sequence↗
PubMed1992

Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli.

We constructed the plasmid pTTB151 in which the E. coli bioB gene was expressed under the control of the tac promoter. Conversion of dethiobiotin to biotin was demonstrated in cell-free extracts of E. coli carrying this plasmid. The requirements for this biotin-forming reaction included fructose-1,6-bisphosphate, Fe2+, S-adenosyl-L-methionine, NADPH, and KCl, as well as dethiobiotin as the substrate. The enzymes were partially purified from cell-free extracts by a procedure involving ammonium sulfate fractionation. Our results suggest that an unidentified enzyme(s) besides the bioB gene product is obligatory for the conversion of dethiobiotin to biotin.

Base Sequence↗