PURIFICATION AND PROPERTIES OF NADPH-DEPENDENT TYLOSIN REDUCTASE FROMSTREPTOMYCES-FRADIAE

A reductase of Streptomyces fradiae was speculated to catalyze reducti on of tylosin to relomycin, an industrially undesirable product. The a ctivity of tylosin reductase was closely related to bacterial growth, suggesting involvement of the enzyme in a primary metabolism. The redu ctase activity was improved significantly in vivo and in vitro. The en zyme was also partially stabilized in vitro. Using a simple five-step chromatographic procedure, the reductase was purified 480-fold to appa rent homogeneity. The purified reductase had a molecular mass of 270 k Da and consisted of two different subunits of 26 and 7 kDa at 1:1 rati o. The enzyme exhibited an absorption maximum at 405 nm and was inhibi ted by exogenous FAD or FMN, indicating a flavin as its prosthetic gro up. Tylosin reductase was optimally active at pH 7.0-7.2 and 40-degree s-C with NADPH as a preferred electron donor. The K(m) of the enzyme f or tylosin was 1.4 mm and that for NADPH was 0.15 mM. The V(max) for t he enzymatic reaction was 917 mumol of tylosin formed/min/mg protein. The enzymatic conversion of tylosin to relomycin was coupled to that o f NADPH to NADP+ at a stoichiometric ratio of 1:1. Tylosin reductase s howed a broad substrate specificity toward all macrolide aldehydes (as normal and shunt metabolites of tylosin biosynthesis) tested. Thus, t he enzyme may have a physiological role of macrolide detoxification fo r the bacterium.