Mechanistic Studies of the Adenosylmethionine Radical Enzyme Biotin Synthase

生物素是一种小分子辅助因子，用于进行涉及羧酸基转移的反应的几种重要酶中；例如，含有生物素的乙酰辅酶a羧化酶在乙酰辅酶a上添加羧酸基团是人类以及大多数其他生物脂肪酸生物合成的第一步。生物素不是在人体中产生的（它是一种必需的维生素），而是在细菌、酵母和植物中通过一种保守的途径产生的，这种途径的结果是一个硫原子取代了两个氢原子，这一反应是由生物素合成酶催化的。这种生化反应是独特的和前所未有的：它需要s -腺苷- l-蛋氨酸（AdoMet或SAM）从前体去硫生物素中去除氢原子，并且它似乎使用铁硫簇辅助因子作为构建生物素的硫源。SAM参与去除氢原子的过程表明，生物素合成酶属于一个新兴的酶家族，它利用SAM作为底物或辅助因子来促进底物或蛋白质自由基的受控生成。在目前的研究中，将使用几种技术来研究这种中间体的形成和衰变。首先，质谱和重原子标记将用于确定中间体的化学性质，探测中间体的形成和衰变动力学，并优化在该状态下酶量最大化的条件。其次，利用振动波谱和电子顺磁共振波谱在同位素标记的底物上探测9-巯基脱硫生物素在催化反应过程中是否共价附着在铁硫簇上。对控制SAM自由基生成、底物激活和碳硫键形成的因素的详细描述将有助于我们了解所有自由基酶共同的机制和结构特征。更广泛的影响。所描述的研究项目为向本科生、研究生和博士后研究人员教授酶反应中间体表征的基本技术提供了一个极好的论坛。夏威夷大学为不同的学生群体提供服务，其中包括来自夏威夷和太平洋岛屿的很大一部分学生，许多本科生将通过参加“定向研究”课程接受蛋白质和酶表征方面的培训。更具体地说，该研究项目将在三年内支持2-3名研究生和3-4名本科生的科学工作。此外，我们对生物素合成酶机制的了解的提高将有助于生物工程过度生产生物素的发展。生物素是一种昂贵但必需的维生素，被纳入人类营养补充剂，更重要的是，被纳入动物饲料。目前的研究可能有助于生产生物素（以及其他维生素）的低成本生物方法，这将通过降低成本和提高粮食生产效率来造福社会。该奖项由分子和细胞生物科学部以及化学部的有机和大分子化学项目共同资助。

Biotin is a small molecular cofactor used in several important enzymes that carry out reactions that involve the transfer of carboxylate groups; for example, the addition of a carboxylate group to acetyl CoA by the biotin-containing enzyme acetyl CoA carboxylase is the first step in fatty acid biosynthesis in humans as well as most other organisms. Biotin is not made in humans (it is an essential vitamin), but is made in bacteria, yeast, and plants by a conserved pathway that concludes with the substitution of a sulfur atom in place of two hydrogen atoms, a reaction that is catalyzed by the enzyme biotin synthase. This biochemical reaction is unique and unprecedented: it requires S-adenosyl-L-methionine (AdoMet or SAM) to remove the hydrogen atoms from the precursor dethiobiotin and it appears to use an iron-sulfur cluster cofactor as the source of sulfur for constructing biotin. The involvement of SAM in removing hydrogen atoms suggests that biotin synthase belongs to an emerging family of enzymes that utilize SAM as a substrate or cofactor to facilitate the controlled generation of substrate or protein radicals. In the present research, the formation and decay of this intermediate will be examined using several techniques. First, mass spectrometry and heavy atom labeling will be used to determine the chemical nature of the intermediate, probe the kinetics of formation and decay of the intermediate, and optimize conditions for maximizing the amount of enzyme in this state. Second, vibrational spectroscopy and electron paramagnetic resonance spectroscopy with isotopically-labeled substrates will be employed to probe whether 9-mercaptodethiobiotin is covalently attached to the iron-sulfur cluster during the catalytic reaction. A detailed description of the factors that control SAM radical generation, substrate activation, and carbon-sulfur bond formation will contribute to our understanding of mechanistic and structural features common to all radical enzymes.BROADER IMPACTS. The research projects described provide an excellent forum for teaching the basic techniques involved in characterizing enzyme reaction intermediates to undergraduate and graduate students and postdoctoral researchers. The University of Hawaii serves a diverse student population that includes a significant percentage of students from the Hawaiian and Pacific Islands, and a number of undergraduates will be trained in protein and enzyme characterization through participation in a "Directed Research" course. More specifically, this research project will support the scientific work of 2-3 graduate students and 3-4 undergraduate students over three years. In addition, our improved knowledge of the mechanism of biotin synthase will contribute to the development of organisms engineered to overproduce biotin. Biotin is an expensive but essential vitamin that is incorporated into both human nutritional supplements and, more significantly, into animal feedstock. The current research could contribute to a low-cost biological method for the production of biotin (as well as other vitamins) which would benefit society by contributing to less expensive and more efficient food production. This award was co-funded by the Division of Molecular and Cellular Biosciences and the Organic and Macromolecular Chemistry Program of the Chemistry Division.