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

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

Research output: Contribution to journalArticle

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 Jan 1

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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