An NADPH:FAD oxidoreductase from the valanimycin producer, Streptomyces viridifaciens. Cloning, analysis, and overexpression.
The valanimycin producer Streptomyces viridifaciens contains a two-component enzyme system that catalyzes the oxidation of isobutylamine to isobutylhydroxylamine. One component of this enzyme system is isobutylamine hydroxylase, and the other component is a flavin reductase. The gene (vlmR) encoding the flavin reductase required by isobutylamine hydroxylase has been cloned from S. viridifaciens by chromosome walking. The gene codes for a protein of 194 amino acids with a calculated mass of 21,265 Da and a calculated pI of 10.2. Overexpression of the vlmR gene in Escherichia coli as an N-terminal His-tag derivative yielded a soluble protein that was purified to homogeneity. Removal of the N-terminal His-tag from the overexpressed protein by thrombin cleavage also produced a soluble protein. Both forms of the protein exhibited a high degree of flavin reductase activity, and the thrombin-cleaved form functioned in combination with isobutylamine hydroxylase to catalyze the conversion of isobutylamine to isobutylhydroxylamine. Kinetic data indicate that the overexpressed protein utilizes FAD and NADPH in preference to FMN, riboflavin, and NADH. The deduced amino acid sequence of the VlmR protein exhibited similarity to several other flavin reductases that may constitute a new family of flavin reductases.
Parry
R J
RJ
Department of Chemistry, Rice University, Houston, Texas 77005-1892, USA.
Li
W
W
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis; D001391:Azo Compounds / Q000138:chemical synthesis; D001426:Bacterial Proteins; D001483:Base Sequence; D016386:Chromosome Walking; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005415:Flavins / Q000378:metabolism; D005798:Genes, Bacterial; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D001391:Azo Compounds; D001426:Bacterial Proteins; D005415:Flavins; D011994:Recombinant Proteins; C048267:valanimycin; D006899:Mixed Function Oxygenases; C103668:VlmH protein, Streptomyces viridifaciens; D009247:NADH, NADPH Oxidoreductases