IDENTIFICATION OF A FLAVIN-NADH OXIDOREDUCTASE INVOLVED IN THE BIOSYNTHESIS OF ACTINORHODIN - PURIFICATION AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

The biosynthesis of the polyketide antibiotic actinorhodin by Streptom yces coelicolor involves the oxidative dimerization and hydroxylation of a precursor, most likely dihydrokalafungin, as the final steps in i ts formation. Mutations in the actVB gene block these last steps, and the mutants secrete kalafungin as a shunt product. To investigate the role of the actVB gene in these transformations, we have overexpressed the gene in Escherichia coli and purified and characterized the recom binant protein. ActVB was shown to catalyze the reduction of FMN by NA DH to give NAD and FMNH(2), which, unusually, is released into solutio n. The protein contains no chromogenic cofactors and exhibits no requi rements for added metal ions. The reaction obeys simple kinetics and p roceeds through the formation of a ternary complex; K-m values for FMN and NADH are 1.5 and 7.3 mu M, respectively, and k(cat) is about 5 s( -1). FAD and riboflavin are also substrates for the enzyme, although t hey have much higher K-m values. The subunit structure of the enzyme w as investigated by analytical ultracentrifugation, which showed the pr otein to exist in rapid equilibrium between monomer and dimer forms. T he possible role of this oxidoreductase in the oxidative chemistry of actinorhodin biosynthesis is discussed.