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 ...

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

SummaryFour pap genes (papA, papB, papC, papM ) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino-L-phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI ¹ phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N-methylation steps of 4-amino-L-phenylalanine leading to DMPAPA via 4-methylamino-L-phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.

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.

TnS Spr transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide:dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P.denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and a-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and a-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.Conversion of cobinamide into cobalamin requires four enzymatic steps in Propionibacterium shermanii (17,24). First, phosphorylation of the hydroxyl group of the (R)-1-amino-2-propanol residue of cobinamide leads to cobinamide phosphate (reaction A). Second, addition of the GMP moiety of a molecule of GTP onto cobinamide phosphate generates GDP-cobinamide (reaction B). Third, exchange of GMP with a-ribazole 5'-phosphate within GDP-cobinamide yields cobalamin 5'-phosphate (reaction C). Fourth, dephosphorylation of cobalamin 5'-phosphate gives cobalamin (reaction D).