Sequence analysis and expression of rubN2 as dTDP-glucose 4,6-dehydratase gene cloned from Streptomyces achromogenes var. rubradiris NRRL3061, rubradirin producer

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Title
Sequence analysis and expression of rubN2 as dTDP-glucose 4,6-dehydratase gene cloned from Streptomyces achromogenes var. rubradiris NRRL3061, rubradirin producer
Author(s)
Jin Chul Yoo; Ji Young Choi; Sung Cheol Yun; Chun Gyu Kim; Jung Joon Lee; Sook Bok Kim; Jae Kyung Sohng
Bibliographic Citation
Journal of Biochemistry Molecular Biology & Biophysics, vol. 4, pp. 313-321
Publication Year
2000
Abstract
Southern blot analysis of the genomic library of Streptomyces achromogenes var. rubradiris NRRL3061, a rubradirin producer revealed the presence of three genes homologous to dTDP-glucose 4,6-dehydratase gene. Only rubN2 is located closely to the locus for polyketide synthase gene and aminohydroxybenzoic acid synthase gene being involved in the biosynthesis of rubransarol moiety of rubradirin. The rubN2 was expressed in Escherichia coli BL21(DE3) using the T7 expression vector pRSET-B. The purified protein was a homodimer with a subunit of molecular weight of 39,000. Only dTDP-glucose was utilized as a substrate by the expressed enzyme. The K(m) for dTDP-glucose and V(max) values were determined to be 35 μM and 199 nmol/min/mg protein, respectively. The K(m) for NAD+ was 20 μM. DNA sequencing of rubN2 and biochemical studies of RubN2 suggest that the gene encode dTDP-glucose 4,6-dehydratase highly specific for dTDP-glucose and obligate biosynthesis of rubranitrose.
Keyword
biosynthesisdTDP-glucose 4,6-dehydrataseNAD+rubranitrosestreptomycesrubradirin
ISSN
I000-0095
Type
Article
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