CLONING AND ANALYSIS OF STRUCTURAL GENES FROM STREPTOMYCES-PRISTINAESPIRALIS ENCODING ENZYMES INVOLVED IN THE CONVERSION OF PRISTINAMYCIN-IIB TO PRISTINAMYCIN-IIA (PIIA) - PIIA SYNTHASE AND NADH-RIBOFLAVIN 5'-PHOSPHATE OXIDOREDUCTASE

In Streptomyces pristinaespiralis, two enzymes are necessary for conve rsion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the maj or component of pristinamycin (D. Thibaut, N. Ratet, D, Bisch, D. Fauc her, L, Debussche, and F. Blanche, J. Bacteriol, 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA a nd SnaB proteins, which catalyzes the oxidation of PIIB to PIIA and th e NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN re ductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes desig ned from limited peptide sequence information of the purified proteins , the corresponding genes were cloned from a genomic library of S. pri stinaespiralis, SnaA and SnaB showed no significant similarity with pr oteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulatio n of PIIB. Complementation of a S. pristinaespiralis PIIA- PIIB+ mutan t with the snaA and snaB genes, cloned in a low-copy-number plasmid, p artially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN re ductases but was 39% identical with the product of the actVB gene of t he actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furth ermore, an S. coelicolor A(3)2 mutant blocked in this step was success fully complemented by the snaC gene, restoring the production of actin orhodin.