The understanding of how living systems "synthesize" the molecules that make them up can provide important information on the relationships among the parts of the plant and animal kingdoms. This can be used in the designing of new plants where the intent is the diminishing/elimination (or conversely increasing/adding) of a constituent. This grant is in the Organic and Macromolecular Chemistry Program. 6-Hydroxyanthranilic acid has been recently identified in this laboratory as a new product of the shikimic acid pathway and as a key intermediate in the biosynthesis of sarubicin A by Streptomyces helicus. 6-Hydroxyanthranilic acid seems most reasonably formed from the rare isochorismic acid. Subsequent steps are proposed involving novel C-glycosides of a 2,6-dideoxy sugar and further hydroxylation of the anthranilate moiety. Subsequent work in this laboratory on the biosynthesis of antibiotic LL-C10037a, with a very different skeleton from sarubicin A, clearly points towards anthranilic acid and/or 3- hydroxyanthranilic acid as a key intermediate with subsequent further hydroxylation at an undetermined stage as well. Up to six putative C-glycoside intermediates will be synthesized with stable isotope labels and tested in S. helicus. Labeled anthranilic acid, 3-hydroxyanthranilic acid, and a series of further hydroxylated and/or decarboxylated and/or acetylated compounds will be used to determine specific biosynthetic incorporations. Cell-free preparations of S. helicus will be generated and examined for the presence of isochorismate synthase and 6-hydroxyanthranilate synthase, and preliminary studies on the mechanism of the former reaction will be initiated with O-18 labels.
