Genetic and biochemical characterization of C-1027 enediyne biosynthesis cluster from Streptomyces globisporus

The enediyne antitumor antibiotics are appreciated for their highly unusual molecular architecture, phenomenal biological activity, and fascinating modes of action. Though the family is structurally diverse all members contain a unit consisting of two acetylenic groups conjugated to a double bond or incipient double bond within a nine or ten-membered ring. This and other unique structural features, coupled with promising clinical potential make the biosynthesis of the enediynes of interest in unveiling new biochemical mechanisms and forwarding combinatorial biosynthesis of novel metabolites through manipulating the genes governing their biosynthesis.; C-1027, an enediyne isolated from Streptomyces globisporus, was selected as a model for enediyne biosynthesis. An approximately 85 kb region of the S. globisporus genome was cloned based on its hybridization with labeled fragments from the genes encoding the C-1027 apoprotein and a well-conserved glucose dehydratase. Characterization of the isolated gene cluster revealed over 55 genes participating in C-1027 biosynthesis including (i) an iterative type I polyketide synthase (PKS) that is distinct from any bacterial PKS known to date, (ii) a general polyketide pathway for the biosynthesis of both the 9 and 10-membered enediyne antibiotics, and (iii) mechanisms for the convergent biosynthesis of the C-1027 chromophore from four building blocks.; Participation of many genes identified in C-1027 biosynthesis was confirmed genetically by the creation of over 25 mutant strains of S. globisporus . These mutants carried inactivate alleles of targeted genes and either lost their ability to produce C-1027 or produced novel analogs of C-1027 as demonstrated by antimicrobial assays and/or HPLC characterization of the isolated chromophore.; In some cases biochemical characterization of a C-1027 gene product revealed novel enzyme mechanisms. SgcC4 is, by homology, a member of a family of histidine/phenylalanine ammonia lyases that employ an unusual 5-methylidene imidazole-5-one (MIO) prosthetic group. The presence of this group was confirmed in SgcC4 by chemical, spectroscopic, and mutational analysis. In contrast with other ammonia lyases, SgcC4 is principally an aminomutase that catalyzes the conversion of L-tyrosine to (S)-β-tyrosine. This finding extends MIO-dependent catalysis from simple ammonia lyases to that of aminomutase and presents an unprecedented mechanism for achieving 1,2-amino rearrangements.