A1 Refereed original research article in a scientific journal
Characterization of aklavinone-11-hydroxylase from Streptomyces purpurascens
Authors: Niemi J, Wang YL, Airas K, Ylihonko K, Hakala J, Mantsala P
Publisher: ELSEVIER SCIENCE BV
Publication year: 1999
Journal: Biochimica et Biophysica Acta: Protein Structure and Molecular Enzymology
Journal name in source: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
Journal acronym: BBA-PROTEIN STRUCT M
Volume: 1430
Issue: 1
First page : 57
Last page: 64
Number of pages: 8
ISSN: 0167-4838
DOI: https://doi.org/10.1016/S0167-4838(98)00265-9
Abstract
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.
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.
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