Mechanistic Studies of the Adenosylmethionine Radical Enzyme Biotin Synthase

腺苷甲硫氨酸自由基酶生物素合酶的机理研究

• 批准号: 0923829
• 负责人: Joseph Jarrett
• 金额: $ 45万
• 依托单位: University of Hawaii
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0923829&HistoricalAwards=false
• 关键词: Mechanistic; Studies; Adenosylmethionine; Radical; Enzyme

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.

生物素是一种小分子辅因子，在几种重要的酶中进行涉及羧酸基转移的反应；例如，在人类和大多数其他生物中，含有生物素的酶乙酰辅酶A羧基酶将羧酸基加成到乙酰辅酶A上是脂肪酸生物合成的第一步。生物素不是在人类中制造的(它是一种必需的维生素)，但在细菌、酵母和植物中是通过一条保守的途径制造的，该途径以硫原子取代两个氢原子结束，该反应由生物素合成酶催化。这种生化反应是独一无二的，也是史无前例的：它需要S-腺苷-L-蛋氨酸(ADOMet或SAM)来去除前体脱硫生物素中的氢原子，而且它似乎使用铁-硫簇辅助因子作为构建生物素的硫源。SAM参与了氢原子的去除，这表明生物素合成酶属于一个新兴的酶家族，它利用SAM作为底物或辅因子来促进底物或蛋白质自由基的可控产生。在本研究中，将使用几种技术来研究这种中间体的形成和衰变。首先，将使用质谱学和重原子标记来确定中间体的化学性质，探索中间体的形成和衰变的动力学，并优化在这种状态下最大化酶的量的条件。其次，将利用同位素标记底物的振动光谱和电子顺磁共振光谱来探测9-巯基脱硫生物素在催化反应过程中是否共价连接到铁-硫簇上。详细描述控制SAM自由基产生、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的共同机理和结构特征。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表征酶反应中间体所涉及的基本技术提供了一个极好的论坛。夏威夷大学服务于多样化的学生群体，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一门“定向研究”课程接受蛋白质和酶特性方面的培训。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更深入的了解将有助于开发出能够过量生产生物素的工程生物。生物素是一种昂贵但必不可少的维生素，它被加入到人类营养补充剂中，更重要的是，被纳入动物饲料中。目前的研究可能有助于开发一种低成本的生物方法来生产生物素(以及其他维生素)，这将有助于降低成本和提高粮食生产效率，从而造福社会。该奖项由分子和细胞生物科学部和化学部的有机和高分子化学项目共同资助。