Mechanistic Studies of the Adenosylmethionine Radical Enzyme Biotin Synthase

生物素是一种小分子辅因子，用于几种重要的酶中，这些酶进行涉及羧酸基团转移的反应;例如，通过含生物素的酶乙酰辅酶A羧化酶将羧酸基团添加到乙酰辅酶A是人类以及大多数其他生物体脂肪酸生物合成的第一步。 生物素不是在人体内产生的（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，该途径以一个硫原子取代两个氢原子结束，这是一种由生物素合酶催化的反应。这种生物化学反应是独特的和前所未有的：它需要S-腺苷-L-甲硫氨酸（SAM）从前体脱硫生物素中去除氢原子，并且它似乎使用铁-硫簇辅因子作为构建生物素的硫源。SAM参与去除氢原子表明，生物素合酶属于新兴的酶家族，其利用SAM作为底物或辅因子以促进底物或蛋白质自由基的受控产生。在本研究中，将使用几种技术来检查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的动力学，并优化条件以使该状态下的酶量最大化。第二，振动光谱和电子顺磁共振光谱与同位素标记的基板将被用来探测9-mercaptodethiobiotin是否共价连接到铁-硫簇在催化反应。详细描述控制SAM自由基生成、底物活化和碳硫键形成的因素将有助于我们理解所有自由基酶共有的机制和结构特征。 所描述的研究项目为本科生、研究生和博士后研究人员教授酶反应中间体表征所涉及的基本技术提供了一个很好的论坛。夏威夷大学为多样化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相当大比例的学生，一些本科生将通过参加“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，我们对生物素合酶机制的进一步了解将有助于开发生物素过量生产的生物体。生物素是一种昂贵但必不可少的维生素，它被掺入人类营养补充剂中，更重要的是，被掺入动物饲料中。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，这将有利于社会，有助于降低成本，提高粮食生产效率。该奖项由分子和细胞生物科学部以及化学部的有机和高分子化学计划共同资助。

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.