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

被引:70
作者
KENDREW, SG
HARDING, SE
HOPWOOD, DA
MARSH, ENG
机构
[1] UNIV CAMBRIDGE,DEPT BIOCHEM,CAMBRIDGE CB2 1QW,ENGLAND
[2] UNIV CAMBRIDGE,CAMBRIDGE CTR MOLEC RECOGNIT,CAMBRIDGE CB2 1QW,ENGLAND
[3] UNIV NOTTINGHAM,DEPT APPL BIOCHEM & FOOD SCI,SUTTON LE12 5RD,LEICS,ENGLAND
[4] JOHN INNES CTR PLANT SCI RES,DEPT GENET,NORWICH NR4 7UH,NORFOLK,ENGLAND
基金
英国惠康基金;
关键词
D O I
10.1074/jbc.270.29.17339
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The biosynthesis of the polyketide antibiotic actinorhodin by Streptomyces coelicolor involves the oxidative dimerization and hydroxylation of a precursor, most likely dihydrokalafungin, as the final steps in its 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 recombinant protein. ActVB was shown to catalyze the reduction of FMN by NADH to give NAD and FMNH(2), which, unusually, is released into solution. The protein contains no chromogenic cofactors and exhibits no requirements for added metal ions. The reaction obeys simple kinetics and proceeds 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 they have much higher K-m values. The subunit structure of the enzyme was investigated by analytical ultracentrifugation, which showed the protein to exist in rapid equilibrium between monomer and dimer forms. The possible role of this oxidoreductase in the oxidative chemistry of actinorhodin biosynthesis is discussed.
引用
收藏
页码:17339 / 17343
页数:5
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