Purification and properties of NADPH-dependent tylosin reductase from Streptomyces fradiae.
A reductase of Streptomyces fradiae was speculated to catalyze reduction of tylosin to relomycin, an industrially undesirable product. The activity of tylosin reductase was closely related to bacterial growth, suggesting involvement of the enzyme in a primary metabolism. The reductase activity was improved significantly in vivo and in vitro. The enzyme was also partially stabilized in vitro. Using a simple five-step chromatographic procedure, the reductase was purified 480-fold to apparent homogeneity. The purified reductase had a molecular mass of 270 kDa and consisted of two different subunits of 26 and 7 kDa at 1:1 ratio. The enzyme exhibited an absorption maximum at 405 nm and was inhibited by exogenous FAD or FMN, indicating a flavin as its prosthetic group. Tylosin reductase was optimally active at pH 7.0-7.2 and 40 degrees C with NADPH as a preferred electron donor. The Km of the enzyme for tylosin was 1.4 mM and that for NADPH was 0.15 mM. The Vmax for the enzymatic reaction was 917 mumol of tylosin formed/min/mg protein. The enzymatic conversion of tylosin to relomycin was coupled to that of NADPH to NADP+ at a stoichiometric ratio of 1:1. Tylosin reductase showed a broad substrate specificity toward all macrolide aldehydes (as normal and shunt metabolites of tylosin biosynthesis) tested. Thus, the enzyme may have a physiological role of macrolide detoxification for the bacterium.
Huang
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Lilly Research Laboratories, Division of Eli Lilly and Company, Indianapolis, Indiana 46285.
Hassell
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TC
Yeh
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D000429:Alcohol Oxidoreductases; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D002845:Chromatography; D004795:Enzyme Stability; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D015645:Tylosin / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D000429:Alcohol Oxidoreductases; C082907:tylosin reductase; D000445:Aldehyde Oxidoreductases; D015645:Tylosin