The application of irreversible enzyme inactivation by """"""""suicide"""""""" substrates to the study of the enzymology of secondary metabolism is proposed. This strategy has as long-term aims the ability to: 1) confirm or refute mechanistic hypotheses for a given biosynthetic transformation; 2) generate auxotrophs at the enzyme level; 3) label specific enzymes of secondary metabolism so as to facilitate identification and ultimately, purification of the target enzymes: 4) deprive a producing organism of an apparent metabolic end-product, thus providing an opportunity to study the organism's need for that metabolite; 5) control the production of toxic secondary metabolites. Since secondary metabolism provides a tremendous array of biologically active natural products (such as antibiotics, antineoplastics, and mycotoxins) their mode of production are of both practical and theoretical interest. The feasibility of this strategy will be investigated in the production of tropolones by Talaromyces stipitatus. The focus of the project will be oxygenation and ring expansion steps. Substrate analogs with strategically-placed difluoromethyl, allenyl, propargyl, and cyanomethyl groups (known to be inhibitory to monooxygenases) will be synthesized and incubated with whole cells or cell-free extracts. If secondary metabolism is affected, biosynthetic intermediates will be purified and identified. Radiolabeled inactivators will be synthesized and employed, and labeled proteins separated and characterized by gel electrophoresis and/or HPLC. If possible, 13C-labeled inactivator will be used, and purified dead enzyme studied by 13C NMR, to determine the nature of the inactivation process. Alternatively, degradation of radiolabeled protein will be followed by chromatographic and electrophoretic characterization of labeled fragments.
