| The pluramycins have gained considerable recognition as highly selective DNA alkylating agents. Unlike simple DNA alkylating agents which react preferentially at guanines found in poly-guanine sequences, the pluramycins act both via reversible DNA intercalation and irreversible alkylation of a guanine residue at specific 5'-PyG-3' motifs, with 5'-CGT sites favored over 5'-TGT sites. Hedamycin and pluramycin A are members of the classical pluramycin class of antitumor antibiotics and consist of a planar 4-H-anthra(1,2- b)pyran chromophore which carries two proximal amino sugars critical for activity, and a distal epoxide-containing side chain required for DNA alkylation. The biosynthetic origin of this unique side chain is unknown but its unusual structure suggests the possibility of an alternative priming of an aromatic PKS. The pluramycins are also clearly distinguished by their unique di-C-glycosidic architecture. Unlike their O-glycoside counterparts, C-glycosides are unaffected by hydrolytic or enzymatic cleavage. Based upon synthetic chemistry precedent, some naturally occurring aryl C-glycosides are presumed to arise via an ortho-O-C rearrangement giving rise to a preferred beta-substitution. However, this biosynthetic postulation has not been tested.; Herein, the first report of the cloning and characterization of the core of the biosynthetic gene clusters encoding for hedamycin (S. griseoruber ) and pluramycin A (S. pluricolorscens) biosynthesis is described. Sequence analysis revealed the type II PKS system along with additional genes that implicate a novel aromatic PKS priming mechanism and the putative C-glycosyltransferase genes. Distinct from previously elucidated loci, a unique feature of these clusters is the utilization of an iterative type I PKS system, Hed/PluT and Hed/PluU, which we propose to be critical for the generation of a hexenoate starter unit to serve as a special primer for subsequent aromatic polyketide biosynthesis. The successful development of genetic manipulation methods in the hedamycin producer, S. griseoruber , was used to establish the significance of both type I and type II PKS genes for pluramycin biosynthesis by constructing specific genetic mutants. This unusual utilization of a type I and II PKS systems for the production of aromatic polyketides is a novel finding in natural product biosynthesis. |
