Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization

Toshiki Furuya, Shusuke Takahashi, Yuichiro Iwasaki, Yoshitaka Ishii, Kuniki Kino, Kotaro Kirimura

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    13 Citations (Scopus)

    Abstract

    Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20°C to 50°C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20°C to 55°C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s-1 at 50°C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

    Original languageEnglish
    Pages (from-to)577-585
    Number of pages9
    JournalJournal of Bioscience and Bioengineering
    Volume99
    Issue number6
    DOIs
    Publication statusPublished - 2005

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    Keywords

    • Desulfurization
    • Dibenzothiophene
    • Ferric reductase
    • Flavin reductase
    • Mycobacterium phlei

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering

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