QUINOENZYMES: BIOGENESIS, STRUCTURE AND FUNCTION

醌酶：生物发生、结构和功能

• 批准号: 6498663
• 负责人: JUDITH P KLINMAN
• 金额: $ 32.8万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498663
• 关键词: Escherichia coli; Saccharomyces; X ray crystallography; active sites; amine oxidase (copper); amine oxidoreductase; cofactor; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; hydroquinones; ligands; metalloenzyme; phenylalanine analog; protein sequence

Quinoproteins constitute a new class of catalysts in which their cofactor is derived from a pre-existing amino acid side chain. Cofactors identified thus far are tryptophan tryptophanylquinone, restricted to prokaryotes, 2,4,5-trihydroxyphenylalanine quinone (TPQ), found to be ubiquitous and lysine tyrosylquinone, restricted to mammalian systems. The latter two cofactors were discovered in the laboratory of the P.I. The family of TPQ enzymes has been demonstrated to catalyze cofactor formation in an auto-catalytic manner, raising the question of a how a single protein structure supports the dual functionalities of cofactor biogenesis and catalytic turnover. The yeast amine oxidase from Hansenula polymorpha (HPAO) is our experimental system of choice, since this protein has been expressed in both S. cerevisiae (for studies of catalytic turnover) and in E. coli (for studies of biogenesis). A crystal structure of the wild type enzyme was solved during the previous granting period and studies of numerous mutant forms will be pursued during the projected period. A series of kinetic and structural studies are planned using both WT and mutant enzymes, with the goal of elucidating the nature of chemical intermediates and rate limiting steps in biogenesis. With regard to catalytic turnover, a new mechanism proposed for the oxidative half reaction will be tested via mutagenesis experiments. Several structural motifs have been identified that involve interaction between residues on opposite subunits of the catalytic dimer; the role of these motifs in biogenesis and turnover will be examined. The physiological function of lysyl oxidase is well defined, whereas that of TPQ-containing molecules in higher eukaryotes remains unknown. Additional studies will involve (i) the investigation of the physiological role of the membrane associated TPQ enzymes in higher eukaryotes, (ii) structure function studies of lysyl oxidase, for comparison to HPAO, and (iii) studies of model compounds for both known and potentially new quinocofactors.

醌蛋白构成了一类新型催化剂，其中其辅因子源自预先存在的氨基酸侧链。迄今为止已鉴定的辅因子有色氨酸色氨酸醌（仅限于原核生物）、2,4,5-三羟基苯丙氨酸醌（TPQ）（被发现普遍存在）和赖氨酸酪氨醌（仅限于哺乳动物系统）。 后两个辅因子是在 P.I. 的实验室中发现的。 TPQ 酶家族已被证明能够以自催化方式催化辅因子形成，这就提出了单一蛋白质结构如何支持辅因子生物发生和催化周转的双重功能的问题。 来自多形汉逊酵母 (HPAO) 的酵母胺氧化酶是我们选择的实验系统，因为该蛋白质已在酿酒酵母（用于催化转化研究）和大肠杆菌（用于生物发生研究）中表达。 在之前的授权期间，野生型酶的晶体结构已得到解决，并且在预计期间将进行多种突变形式的研究。 计划使用野生型和突变酶进行一系列动力学和结构研究，目的是阐明化学中间体的性质和生物发生中的限速步骤。 关于催化周转，将通过诱变实验测试为氧化半反应提出的新机制。 已经鉴定出几个结构基序，这些基序涉及催化二聚体相对亚基上的残基之间的相互作用；将检查这些基序在生物发生和周转中的作用。 赖氨酰氧化酶的生理功能已明确，而高等真核生物中含有 TPQ 的分子的生理功能仍不清楚。其他研究将涉及 (i) 高等真核生物中膜相关 TPQ 酶的生理作用研究，(ii) 赖氨酰氧化酶的结构功能研究，以与 HPAO 进行比较，以及 (iii) 已知和已知模型化合物的研究潜在的新藜基因子。