PURIFICATION OF PEPTIDE SYNTHETASES INVOLVED IN PRISTINAMYCIN-I BIOSYNTHESIS

Several assays of pristinamycin I synthetases based on adenylate or th ioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristina espiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating th e first pristinamycin I residue, was purified 200-fold, using an ATP-p yrophosphate exchange assay. This enzyme was shown to be a monomer wit h an M(r) of 67,000 as estimated by sodium dodecyl sulfate polyacrylam ide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with M(r)s of 240,000 and able to bind c ovalently L-threonine as a thioester, was purified 100-fold. This prot ein 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 estim ated to be between 250,000 and 350,000, was purified 200-fold. This la rge enzyme catalyzed thioesterification and subsequent N-methylation o f 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 th e last two pristinamycin I precursors. Finally, a single polypeptide c hain (SnbE) with an M(r) of 170,000, catalyzing L-phenylglycine depend ent ATP-pyrophosphate exchange, was purified 3,000-fold and characteri zed. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four is olated proteins. The purified SnbE protein was further shown to be a p roteolytic fragment of SnbD.