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High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 188.8.131.52.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.