Biosynthesis of PF1022A and related cyclooctadepsipeptides

PF1022A belongs to a recently identified class of N-methylated cyclooctadep sipeptides (CODPs) with strong anthelmintic properties. Described here is t he 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 precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methioni ne in the presence of ATP and MgCl2, A 350-kDa depsipeptide synthetase, PFS YN, responsible for PF1022A synthesis was purified to electrophoretic homog eneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechan ism in which the substrates are activated as thioesters via adenylation, N- Methylation of the substrate L-leucine takes place after covalent binding p rior to peptide bond formation. The enzyme is capable of synthesizing all k nown natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) dif fering in the content of D-lactate and D-phenyllactate. In addition to PF10 22 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of D-lactate and N-methyl-L-leucine), as well as di-, tetra-, an d hexa-PF1022 homologs, PFSYN strongly resembles the well documented enniat in synthetase in size and mechanism. Our results suggest that PFSYN, like e nniatin synthetase, is an enzyme with two peptide synthetase domains and fo rms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of the D-hydroxy acid binding site, D-lactate or D-phenyll actate can be incorporated into the dipeptidols depending on the concentrat ion of these substrates in the reaction mixture.