Purification of peptide synthetases involved in pristinamycin I biosynthesis

Abstract

Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an M r of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with M r s of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein 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 estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 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 the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an M r of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.

Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517

“…PyrA (543 aa), upstream of the NRPS/PKS/NRPS locus, is a probable 3-HPA:AMP ligase because it is similar to SnbA involved in pristinamycin I biosynthesis by S. pristinaespiralis (68% identity) and VisB involved in virginamycin S biosynthesis by S. virginiae (65% identity) (14,26). Presumably, 3-HPA is activated by PyrA and incorporated into the pyridomycin assembly line (Fig.…”

Substrate Specificity of the Nonribosomal Peptide Synthetase PvdD fromPseudomonas aeruginosa

“…Residue 331 (GrsA numbering) was considered to be a coding residue by Stachelhaus et al (38) but not by Challis et al (5). The predicted coding residues of three other L-threonine-activating A domains are from SyrB, a syringomycin synthetase from Pseudomonas syringae (15); AcmB, an actinomycin synthetase from Streptomyces chrysomallus (34); and SnbC, a pristinamycin synthetase from Streptomyces pristinaespiralis (42). Threonine activation was demonstrated by ATP-PP i exchange for SnbC and SyrB.…”

Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species

“…The structural dissimilarity of the type A and type B streptogramins, however, implies that they are biosynthesized by quite different catalytic machinery. Indeed analysis of the genes that direct production of the pristinamycin type I and type II components indicates that they are biosynthesized by nonribosomal peptide and predominantly polyketide pathways, respectively (30)(31)(32)(33). It is therefore hard to imagine how the pathways to the type I and type II metabolites could have evolved from a common origin.…”

“…2)] are assembled through a mixed polyketide͞nonribosomal peptide pathway, whereas the type B streptogramins [e.g., pristinamycin IA (5) (Fig. 2) (R ϭ NMe 2 ) and virginiamycin S 1 (6) (R ϭ H)] are nonribosomally synthesized depsipeptides that contain several unusual nonproteinogenic amino acids (26)(27)(28)(29)(30)(31)(32)(33). 4 is coproduced with 5 by Streptomyces pristinaespiralis NRRL 2958 and with 6 by Streptomyces virginiae (34).…”