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

Abstract

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 ...

Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia coli W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

“…The K m values for NADH and different flavins (Table 1) were similar to those observed for other flavin reductases, that is, enzymes that generate free reduced flavins (10,18,21,38). It is worth noting the high K m values for FMN when compared to the K m values in the nanomolar range that have been determined for other monooxygenases, an observation that reinforces the idea that FMN acts as a substrate rather than as a prosthetic group.…”

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4‐dimethylamino‐l‐phenylalanine precursor of pristinamycin I

“…Liquid cultures for pristinamycin production were made as described by Thibaut et al (1995). Nosiheptide (for selection of thiostrepton resistance; Rhô nePoulenc Rorer) was used at a concentration of 400 g ml ¹1 for solid media and 2 g ml ¹1 for liquid media.…”

“…Extraction, HPLC separation and quantification of PI and PII components PI and PII are extracted and analysed by high-performance liquid chromatography (HPLC) as previously described (Thibaut et al, 1995). Low PI concentrations were detected with a Kratos Spectroflow 980 fluorescence detector set at ᮊ 1997 Blackwell Science Ltd, Molecular Microbiology,23,[191][192][193][194][195][196][197][198][199][200][201][202] Expression of PapM in E. coli A 1.7 kb MluI-StuI fragment, harbouring the end of the papB gene and the entire papM gene, was first cloned into pMTL23, linearized by MluI and BamHI and then treated with Klenow to give pVRC706, in which the BamHI site was reconstituted.…”

“…The previously described cell-lysis procedure ( Thibaut et al, 1995) was improved as follows to avoid protein degradation. 1 mM PMSF, 5 mM EDTA, 5 mM EGTA and 500 mM KCl were included in the washing buffer.…”

Reduced Flavins Promote Oxidative DNA Damage in Non-respiringEscherichia coli by Delivering Electrons to Intracellular Free Iron

“…Whereas FADH 2 most commonly serves as a redox cofactor when it is tightly bound to enzymes, free FADH 2 is used as a co-substrate for monooxygenase reactions (42)(43)(44)(45)(46), including several from E. coli strains (28,47,48). Indirect evidence has suggested that FADH 2 may also assist in the activation of ribonucleotide reductase (33).…”