SUICIDE SUBSTRATES IN SECONDARY METABOLISM

不可逆酶失活在“自杀”中的应用 次生代谢物酶的底物研究 新陈代谢被提出。这一战略的长期目标是 能够：1)确认或反驳机械假说 给出了生物合成转化；2)在 酶水平；3)标记次生代谢特异性酶 从而便于鉴定并最终提纯 目标酶：剥夺生产有机体的 明显的代谢最终产物，从而提供了一个机会 研究生物体对该代谢物的需求；5)控制 产生有毒的次生代谢物。 由于次级新陈代谢提供了大量的 具有生物活性的天然产物(如抗生素、 抗肿瘤和真菌毒素)，它们的生产方式是 既有实践兴趣，也有理论兴趣。这样做的可行性 将通过以下方式研究特罗隆的生产策略 枯草芽孢杆菌。该项目的重点将是 充氧和扩环步骤。底物类似于 二氟甲基，烯丙基，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培养 萃取物。如果次生代谢受到影响，生物合成 中间体将被提纯和鉴定。无线电标记的 灭活剂将被合成和使用，并标记 凝胶电泳法分离和鉴定蛋白质 和/或高效液相色谱仪。如果可能，将使用13C标记的灭活剂， 并用~(13)C核磁共振仪对纯化的死酶进行了研究，以确定 失活过程的性质。或者，降级 放射性标记的蛋白质将随后进行层析和 标记片段的电泳学表征。

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.