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.
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. 1.6.8.1.) (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.
Thibaut
D
D
Département Analyse, Centre de Recherche de Vitry-Alfortville, Rhône-Poulenc Rorer S.A., Vitry-sur-Seine, France.
Ratet
N
N
Bisch
D
D
Faucher
D
D
Debussche
L
L
Blanche
F
F
D000595:Amino Acid Sequence; D038181:FMN Reductase; D005486:Flavin Mononucleotide / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010101:Oxygen Consumption; D010105:Oxygenases / Q000032:analysis / Q000235:genetics*; D011392:Proline / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D014769:Virginiamycin / Q000096:biosynthesis* / Q000378:metabolism
D009243:NAD; D014769:Virginiamycin; D005486:Flavin Mononucleotide; D011392:Proline; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D010105:Oxygenases; C095499:pristinamycin IIA synthase; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases