Biosynthesis of PF1022A and Related Cyclooctadepsipeptides

Abstract

PF1022A belongs to a recently identified class of Fig. 1. Structurally, the PF1022 anthelmintics are related to bassianolide, which is an insecticidal metabolite from the fungus Beauveria bassiana (5), and to the enniatins, a class of N-methylated cyclohexadepsipeptides produced by Fusarium species with manifold biological activities (6, 7).Enniatins consist of alternating residues of D-HIV and Nmethylated branched chain amino acids like L-valine or Lisoleucine. Enniatins are synthesized by the multifunctional enzyme ESYN, which was the first N-methyl-cyclopeptide synthetase ever characterized. Biochemical investigations revealed that this enzyme is one single polypeptide chain with a molecular mass of about 350 kDa (8, 9). All catalytic functions necessary for enniatin synthesis, i.e. ATP-dependent substrate activation, thioacylation reactions, N-methylation, and condensation of the covalently bound substrates, are catalyzed by ESYN in a so-called thiotemplate mechanism (10). ESYN represents a hybrid system of a peptide synthetase and a methyltransferase and serves as a model system for other N-methylcyclopeptide synthetases like cyclosporine synthetase (11) and for the peptide synthetase responsible for the synthesis of PF1022 compounds.ESYN catalyzes synthesis of the enniatin molecule from its primary precursors D-HIV and a branched chain amino acid like L-valine or L-isoleucine in an ATP-dependent reaction; the methyl group in the N-methylated peptides is donated by AdoMet. As in other peptide synthetases, 4Јphosphopantethe-ine, covalently attached to the enzyme, acts as a prosthetic group in acylation reactions. Analysis of the ESYN gene (esyn1) of Fusarium scirpi revealed an open reading frame of 9393 base pairs encoding a 347-kDa polypeptide, which contains two highly conserved peptide synthetase domains, EA and EB (9). The EA domain of the protein N-terminal region was identified as the D-HIV binding site. The EB domain of the C-terminal region was identified as the L-amino acid binding site. The EB domain is interrupted by a 434-amino acid insertion responsible for the N-methyltransferase function. An analogous insertion is found in cyclosporine synthetase (9, 12). Both domains (EA and EB) carry a phosphopantetheine prosthetic group.

Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

“…84 This has been confirmed for all other CODSs studied to date, 61,79 and can also be concluded independently from their identical domain organization. Substrate requirements reveal the overall reaction 3 AA + 3 HA + 3 SAM + 6 ATP / COD + 6 AMP + 6 PP i + 3 SAH (1) where…”

“…The naturally found product distributions in fermentations could be reproduced in vitro with the natural substrates. 61 Since the observed substrate spectrum of RsPFSYN includes two sterically and electronically very different hydroxycarboxylic acids, D-Lac and D-PheLac, a certain substrate promiscuity was expected from this enzyme. Various hydroxycarboxylic acids were synthesized and tested with RsPFSYN for the enzymatic assembly of PF1022 analogs, with the range of synthetic substrates extended to >40 aromatic and aliphatic hydroxycarboxylic acids.…”

“…90 Using the enniatin synthetase as a model, the enzymatic reconstitution of the 350 kDa PF1022 synthetase was also achieved, and PF1022 congeners were produced in vitro with the substrates L-Leu, D-Lac, D-PheLac, Mg 2+ , SAM and ATP. 61 The K m values for product formation were determined to be…”

Structural analysis of a non-ribosomal halogenated cyclic peptide and its putative operon from Microcystis: implications for evolution of cyanopeptolins

“…Since N-methylation has been shown to occur on the amino acid monomer prior to peptide bond formation (Billich & Zocher, 1987;Weckwerth et al, 2000), the presence of an NMT-domain in module 6 indicates that the amino acid activated and condensed by this module becomes methylated.…”

Effects of the anthelmintic drug PF1022A on mammalian tissue and cells

“…isolated from the plant Camellia japonica [5]. So far, the nonribosomal N-methylcyclooctadepsipeptide synthetase responsible for PF1022A biosynthesis has been isolated and characterized [11]. Consequently, this discovery allows the development of new N-methylcyclooctadepsipeptide-derived compounds [12].…”