l-683590 and pipecolic-acid

Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Cloning, Molecular; DNA, Bacterial; Fourier Analysis; Genes, Bacterial; Immunosuppressive Agents; Kinetics; Mass Spectrometry; Molecular Structure; Multigene Family; Mutagenesis, Site-Directed; Peptide Synthases; Pipecolic Acids; Recombinant Proteins; Sirolimus; Streptomyces; Substrate Specificity; Tacrolimus

Immunomycin, an immunosuppressant closely related to FK 506, contains a pipecolate residue in amide linkage with an acyl group in its polyketide backbone. An enzyme activating L-pipecolic acid has been isolated from Streptomyces hygroscopicus var. ascomyceticus, which produces immunomycin. Purification results in a monomer of 170 kDa exhibiting N-terminal heterogeneity, apparently arising from proteolysis of a single species. It is a dimer under native conditions. The reaction appears to use an aminoacyl adenylate as an intermediate in the activating reaction, as do most activating enzymes involved in nonribosomal peptide synthesis. A range of pipecolate and proline analogues act as substrates in the pyrophosphate-ATP exchange resulting from the adenylation reaction. Several analogues are inhibitors of the subsequent thioesterification of the enzyme. Antibody raised to the purified enzyme was used to follow antigen during the course of fermentation. Maximal levels of antigen are found when synthesis of immunomycin is maximal. Ten of twelve immunomycin nonproducing mutants lack detectable pipecolate-activating enzyme in Western blots. From the enzymatic characteristics, substrate specificity, and immunological properties, we propose that we have isolated the enzyme responsible for activating pipecolic acid for immunomycin biosynthesis.

Topics: Adenosine Triphosphate; Anti-Bacterial Agents; Bacterial Proteins; Blotting, Western; Heterocyclic Compounds; Immunosuppressive Agents; Molecular Weight; Peptide Synthases; Pipecolic Acids; Streptomyces; Substrate Specificity; Sulfides; Tacrolimus