SUICIDE SUBSTRATES IN SECONDARY METABOLISM

"自杀"不可逆酶失活的应用 酶的研究底物 提出了代谢。 这一战略的长期目标是， 能力：1）证实或反驳机械假说， 给定的生物合成转化; 2）在 酶水平; 3）标记次级代谢的特异酶 从而有助于鉴定并最终纯化 目标酶：4）剥夺生产生物体的 表观代谢终产物，从而提供了机会， 研究生物体对代谢物的需求; 5）控制 产生有毒的次级代谢物。 由于次级代谢提供了大量的 生物活性天然产物（如抗生素， 抗真菌塑料和霉菌毒素），其生产方式是 实践和理论的兴趣。 的可行性 将通过以下方式研究环庚三烯酚酮生产中的策略： 踝节菌 该项目的重点将是 氧化和扩环步骤。 底物类似物， 战略性放置的二氟甲基，丙二烯基，炔丙基，和 氰甲基（已知对单加氧酶有抑制作用） 将被合成并与全细胞或无细胞的 萃取物 如果次级代谢受到影响， 中间体将被纯化和鉴定。 放射标记 将合成和使用灭活剂，并标记 通过凝胶电泳分离和表征的蛋白质 和/或HPLC。 如果可能，将使用13C标记的灭活剂， 并通过13C NMR研究纯化的死酶，以确定 灭活过程的性质。 或者，降解 放射性标记的蛋白将随后进行色谱分析， 标记片段的电泳表征。

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.