ENZYME CATALYSIS AND REGULATION
Biosynthesis of PF1022A and Related Cyclooctadepsipeptides*

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PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursorsl-leucine, d-lactate,d-phenyllactate, and S-adenosyl-l-methionine in the presence of ATP and MgCl2. A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate l-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of d-lactate andd-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of d-lactate andN-methyl-l-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of thed-hydroxy acid binding site, d-lactate ord-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture.

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Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M001084200

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This work was supported by Bayer AG Leverkusen and Meiji Seika Kaisha Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Current address: Max Planck Institute of Molecular Plant Physiology, 14476 Golm, Germany.