High levels of conversion of 14 C-labelled pristinamycin II B (PII B ) to pristinamycin II A (PII A ) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PII B was an intermediate on the pathway to PII A . In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PII B to PII A 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 PII A 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. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH 2 ) to the more abundant second enzyme. The latter was purified only 160-fold and was called PII A synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PII B by molecular oxygen immediately followed by a dehydration leading to PII A . The native PII A synthase consists of two different subunits with M r s 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 M r 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.Pristinamycins I and pristinamycins II (PII), the two families of bacteriostatic components associated in the bactericidal antibiotic pristinamycin, are produced by Streptomyces pristinaespiralis (22,23). Pristinamycins I are cyclohexadepsipeptides belonging to the B group of streptogramins, while PII are polyunsaturated macrolactones of the A group of streptogramins (Fig. 1). Both streptogramins A and B are secondary metabolites synthesized by a large number of different Streptomycetes, and most of the members of these families have received several names, often related to the species from which they were isolated (6, 28). Thus, the main component of PII, pristinamycin II A (PII A ), is also called mikamycin A, ostreogrycin A, staphylomycin M1, streptogramin A, synergistin A, vernamycin A, virginiamycin M1, and PA-114 A1, while the minor component pristinamycin II B (PII B ) is also known as ostreogrycin G and virginiamycin M2 (7).Pristinamycin is poorly water soluble, and its therapeutic use has remained rather restricted until now. The development of a water-soluble streptogramin, RP 59500, has recently revived interest in this old family of antibiotics (1). Pristinamycin is active against both gram-positive bacteria, especially Staphylococcus and Streptococcus spp., and some gram-negative bacteria, such as Haemophilus spp. Interestingly, both ...

Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an M r of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with M r s of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein also activated L-aminobutyric acid, which is further epimerized to generate the third residue of the pristinamycin I macrocycle. A third protein, consisting of two identical subunits (SnbD) with M r s estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 4-dimethylamino-L-phenylalanine, the fifth pristinamycin I residue. SnbD could also activate L-proline, the fourth pristinamycin I residue, and some preparations retained a low but significant activity for the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an M r of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.