Characterization of aklavinone-11-hydroxylase from Streptomyces purpurascens

: Niemi J, Wang YL, Airas K, Ylihonko K, Hakala J, Mantsala P

Publisher: ELSEVIER SCIENCE BV

: 1999

: Biochimica et Biophysica Acta: Protein Structure and Molecular Enzymology

: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY

: BBA-PROTEIN STRUCT M

: 1430

: 1

: 57

: 64

: 8

: 0167-4838

DOI: https://doi.org/10.1016/S0167-4838(98)00265-9

Aklavinone-11-hydroxylase (RdmE) is a FAD monooxygenase participating in the biosynthesis of daunorubicin, doxorubicin and rhodomycins. The rdmE gene encodes an enzyme of 535 amino acids. The sequence of the Streptomyces purpurascens enzyme is similar to other Streptomyces aromatic polyketide hydroxylases. We overexpressed the gene in Streptomyces lividans and purified aklavinone-11-hydroxylase to apparent homogeneity with four chromatographic steps utilizing a kinetic photometric enzyme assay. The enzyme is active as the monomer with a molecular mass of 60 kDa; it hydroxylates aklavinone and other anthracyclinones. Aklavinone-11-hydroxylase can use both NADH and NADPH as coenzyme but it is slowly inactivated in the presence of NADH. The apparent K-m for NADPH is 2 mM and for aklavinone 10 mu M. The enzyme is inactivated in the presence of phenylglyoxal and 2,3-butanedione. NADPH protects against inactivation of aklavinone-11-hydroxylase by phenylglyoxal. (C) 1999 Elsevier Science B.V. All rights reserved.