Characterization of aklavinone-11-hydroxylase from Streptomyces purpurascens

Aklavinone-11-hydroxylase (RdmE) is a FAD monooxygenase participating in th e biosynthesis of daunorubicin, doxorubicin and rhodomycins. The rdmE gene encodes an enzyme of 535 amino acids. The sequence of the Streptomyces purp urascens enzyme is similar to other Streptomyces aromatic polyketide hydrox ylases. We overexpressed the gene in Streptomyces lividans and purified akl avinone-11-hydroxylase to apparent homogeneity with four chromatographic st eps utilizing a kinetic photometric enzyme assay. The enzyme is active as t he monomer with a molecular mass of 60 kDa; it hydroxylates aklavinone and other anthracyclinones. Aklavinone-11-hydroxylase can use both NADH and NAD PH as coenzyme but it is slowly inactivated in the presence of NADH. The ap parent K-m for NADPH is 2 mM and for aklavinone 10 mu M. The enzyme is inac tivated in the presence of phenylglyoxal and 2,3-butanedione. NADPH protect s against inactivation of aklavinone-11-hydroxylase by phenylglyoxal. (C) 1 999 Elsevier Science B.V. All rights reserved.