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 conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D. Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and the NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin.
Blanc
V
V
Unité de Biologie Moléculaire, Centre de Recherche de Vitry-Alfortville, Rhône-Poulenc Rorer S.A., Vitry-sur-Seine, France.
Lagneaux
D
D
Didier
P
P
Gil
P
P
Lacroix
P
P
Crouzet
J
J
D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D038181:FMN Reductase; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D010088:Oxidoreductases; D010105:Oxygenases / Q000032:analysis / Q000235:genetics*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014769:Virginiamycin / Q000096:biosynthesis*; D000095622:Benzoisochromanequinones
D000880:Anthraquinones; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D010088:Oxidoreductases; D010105:Oxygenases; D014769:Virginiamycin; C095499:pristinamycin IIA synthase; C013390:actinorhodin; D000095622:Benzoisochromanequinones