PURIFICATION AND CHARACTERIZATION OF ISOBUTYLAMINE N-HYDROXYLASE FROMTHE VALANIMYCIN PRODUCER STREPTOMYCES-VIRIDIFACIENS MG456-HF10

Streptomyces viridifaciens MG456-hF10 produces the antitumor agent val animycin, which is a member of a family of antibiotics containing the azoxy group, An enzyme involved in the biosynthesis of valanimycin has been purified 360-fold from S. viridifaciens. This enzyme, isobutylam ine N-hydroxylase, catalyzes the oxidation of isobutylamine to isobuty lhydroxylamine in the presence of oxygen and a reduced flavin cofactor , Unlike other known N-hydroxylases, isobutylamine N-hydroxylase canno t carry out the reduction of the flavin cofactor, Rather, the reduced flavin is supplied by a separate flavin reductase that is present in e xtracts of S, viridifaciens, The reduced flavin cofactor could also be supplied by the flavin mononucleotide reductase of Vibro fischeri, Th e requirement for molecular oxygen and a reduced flavin indicates that the N-hydroxylase is a flavin monooxygenase and that the mechanism fo r the hydroxylation is likely to proceed via the formation of a flavin 4a-hydroperoxide, Isobutylamine N-hydroxylase exhibited a subunit mol ecular mass of 40 kDa and existed in dimeric or trimeric form dependin g upon buffer conditions, The pi of the protein was found to be ca, 5. 1 and the enzyme exhibited a sensitivity to thiol-directed reagents. ( C) 1997 Academic Press.