sirolimus and pipecolic-acid

Macrolide-pipecolate natural products, such as rapamycin (1) and FK-506 (2), are renowned modulators of FK506-binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB-M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A-D (4-7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L-lysine into the L-pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP-binding metabolites and for probing new enzymology in nature's polyketide tailoring arsenal.

Topics: Actinomycetales; Amino Acid Sequence; Ammonia-Lyases; Biocatalysis; Cloning, Molecular; Computational Biology; Furans; Molecular Sequence Data; Multigene Family; Pipecolic Acids; Sirolimus; Tacrolimus

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.

Topics: Amino Acid Sequence; Ammonia-Lyases; Enzyme Inhibitors; Escherichia coli; Kinetics; Molecular Sequence Data; Nipecotic Acids; Pipecolic Acids; Recombinant Proteins; Sirolimus

The natural product rapamycin, produced during fermentation by Streptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acid l-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusual l-pipecolate analogs in place of the normal amino acid. Our results suggest that the l-pipecolate analog (+/-)-nipecotic acid inhibits the biosynthesis of l-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (+/-)-nipecotic acid in our precursor-directed biosynthesis studies to reduce l-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.

Topics: Biotechnology; Gene Expression Regulation, Bacterial; Nipecotic Acids; Pipecolic Acids; Protein Precursors; Sirolimus; Streptomyces; Tacrolimus Binding Protein 1A

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

Analysis of the gene cluster from Streptomyces hygroscopicus that governs the biosynthesis of the polyketide immuno-suppressant rapamycin (Rp) has revealed that it contains three exceptionally large open reading frames (ORFs) encoding the modular polyketide synthase (PKS). Between two of these lies a fourth gene (rapP) encoding a pipecolate-incorporating enzyme that probably also catalyzes closure of the macrolide ring. On either side of these very large genes are ranged a total of 22 further ORFs before the limits of the cluster are reached, as judged by the identification of genes clearly encoding unrelated activities. Several of these ORFs appear to encode enzymes that would be required for Rp biosynthesis. These include two cytochrome P-450 monooxygenases (P450s), designated RapJ and RapN, an associated ferredoxin (Fd) RapO, and three potential SAM-dependent O-methyltransferases (MTases), RapI, RapM and RapQ. All of these are likely to be involved in 'late' modification of the macrocycle. The cluster also contains a novel gene (rapL) whose product is proposed to catalyze the formation of the Rp precursor, L-pipecolate, through the cyclodeamination of L-lysine. Adjacent genes have putative roles in Rp regulation and export. The codon usage of the PKS biosynthetic genes is markedly different from that of the flanking genes of the cluster.

Topics: Amino Acid Sequence; Codon; Genes, Bacterial; Molecular Sequence Data; Multienzyme Complexes; Operon; Pipecolic Acids; Polyenes; Sequence Alignment; Sequence Homology, Amino Acid; Sirolimus; Streptomyces

In a chemically defined medium containing aspartate, arginie and histidine to support good growth, addition of L-lysine stimulated rapamycin production by 150%. This was probably due to its conversion to pipecolic acid, a rapamycin precursor. Phenylalanine and methionine interfered in rapamycin production by unknown mechanisms.

Topics: Amino Acids; Antifungal Agents; Culture Media; Lysine; Methionine; Phenylalanine; Pipecolic Acids; Polyenes; Quaternary Ammonium Compounds; Sirolimus; Streptomyces