Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization.
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 degrees C to 50 degrees 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 degrees C to 55 degrees 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 degrees 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.
Furuya
Toshiki
T
Department of Applied Chemistry, School of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Takahashi
Shusuke
S
Iwasaki
Yuichiro
Y
Ishii
Yoshitaka
Y
Kino
Kuniki
K
Kirimura
Kohtaro
K
D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D038181:FMN Reductase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009168:Mycobacterium phlei / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013457:Sulfur Compounds / Q000378:metabolism*; D013696:Temperature; D013876:Thiophenes / Q000378:metabolism*
D013457:Sulfur Compounds; D013876:Thiophenes; D038181:FMN Reductase; C016366:dibenzothiophene