AN NADPH-FAD OXIDOREDUCTASE FROM THE VALANIMYCIN PRODUCER, STREPTOMYCES-VIRIDIFACIENS - CLONING, ANALYSIS, AND OVEREXPRESSION

The valanimycin producer Streptomyces uilidifaciens contains a two-com ponent enzyme system that catalyzes the oxidation of isobutylamine to isobutylhydroxylamine, One component of this enzyme system is isobutyl amine hydroxylase, and the other component is a flavin reductase, The gene (vlmR) encoding the flavin reductase required by isobutylamine hy droxylase has been cloned from S. viridifaciens by chromosome walking, The gene codes for a protein of 194 amino acids with a calculated mas s 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-t erminal His-tag from the overexpressed protein by thrombin cleavage al so produced a soluble protein, Both forms of the protein exhibited a h igh 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 pre ference to FMN, riboflavin, and NADH. The deduced amino acid sequence of the VlmR protein exhibited similarity to several other flavin reduc tases that may constitute a new family of flavin reductases.