PURIFICATION OF THE 2-ENZYME SYSTEM CATALYZING THE OXIDATION OF THE D-PROLINE RESIDUE OF PRISTINAMYCIN-IIB DURING THE LAST STEP OF PRISTINAMYCIN-IIA BIOSYNTHESIS

High levels of conversion of C-14-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristi naespiralis and in some other streptogramin A producers. This establis hed 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, ri boflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were sh own to be necessary to;catalyze this reaction. Both were purified to h omogeneity from S. pristinaespiralis by a coupled enzyme assay based o n the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure inclu ding a decisive affinity chromatography step on FMN-agarose. It was sh own to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH(2)) to the more abundant s econd enzyme. The latter was purified only 160-fold and was called PII A synthase. Our data strongly suggest that this enzyme catalyzes a tra nsient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with M(r)s of around 50,000 and 35,000, as est imated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, w hile the FMN reductase seems to be a monomer with a M(r) of around 28, 000 and containing one molecule of tightly,bound FMN. Stepwise Edman d egradation of the entire polypeptides or some of their trypsin-digeste d fragments provided amino acid sequences for the two isolated protein s.