Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis.

摘要: High levels of conversion 14C-labelled pristinamycin IIB (PIIB) to IIA (PIIA) were obtained in vivo Streptomyces pristinaespiralis and some other streptogramin A producers. This established that PIIB was an intermediate on the pathway PIIA. In addition, vitro studies with cell-free protein preparations demonstrated oxidation PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), molecular oxygen. Two enzymes shown be necessary catalyze this reaction. Both purified homogeneity from S. by coupled enzyme assay based formation addition complementing enzyme. One about 3,000-fold procedure including decisive affinity chromatography step FMN-agarose. It NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced (FMNH2) more abundant second The latter only 160-fold synthase. Our data strongly suggest catalyzes transient hydroxylation oxygen immediately followed dehydration leading native synthase consists two different subunits Mrs around 50,000 35,000, as estimated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while reductase seems monomer Mr 28,000 containing one molecule tightly bound FMN. Stepwise Edman degradation entire polypeptides or their trypsin-digested fragments provided amino acid sequences for isolated proteins.