Functional Characterization Of MarF In Biosynthetic Gene Cluster Of Diketopiperazine Indole Alkaloid Maremycin

Due to their simple core structure and their potent biological properties,diketopiperazines became a valuable lead structure for drug design.The functional information of their biosynthetic genes will provide opportunities for these structural modifications by manipulating their biosynthetic pathways.Thus,the subject of this research is the biosynthesis of an indole alkaloid maremycin with diketopiperazine core from marine-derived Streptomyces sp.B9173.The role of marF from this gene cluster was elucidated using techniques including molecular genetics,natural product chemistry and enzymology biochemistry.Six new N-demethylmaremycins(11-16)were obtained from the mutant with marF inactivated.Their planar structures were elucidated by MS and NMR spectroscopy.The absolute configurations of 11 and 14 were determined by X-ray diffraction analysis,in particular for an unexpected D-tryptophan unit,and the C-3 is determined as R-configuration.Furthermore,the absolute configurations of 12,13,15,and 16 were deduced from NOESY and CD spectra.The accumilation of these compounds indicated that marF encodes an N-methyltransferase responsible for the methylation of the indole-nitrogen in the L-tryptophan moiety of the maremycins.Biochemical analysis of MarF in vitro proved that N-methylation on indole is not the final step in biosynthesis of MARs and probably takes place at the early stages of the biosynthesis of MARs.A plausible explanation for the tryptophan epimerization in the N-demethylmaremycin biosynthesis is also provided.The functional elucidation of gene marF and discovery of N-demethylmaremycins with D-tryptophan unit enriched the diversities in methylation and epimerization of diketopiperazines for research.