Mechanistic Studies of the S-Adenosylmethionine Radical Enzyme Biotin Synthase

智力优势：酶可以利用自由基的高反应性来进行其他困难的化学转化。这种酶存在于生命的所有分支中，特别是在各种微生物中普遍存在。在几乎所有生物体中发现的一个新兴的酶超家族利用S-腺苷-L-甲硫氨酸（S-腺苷-L-蛋氨酸或SAM）作为底物或辅因子来产生底物或蛋白质自由基。自由基SAM超家族的一个重要子集催化自由基介导的硫向生物分子的加成。生物素合酶催化硫氧化加成到脱硫生物素上的饱和碳原子上，产生生物素硫番环。生物素合酶是一种铁硫酶，同位素标记表明掺入生物素的硫原子来自铁硫簇。我们的研究重点是了解自由基产生、底物活化和硫掺入的化学细节。在生物素合酶反应序列的工作假设中，以与所有自由基SAM酶相同的方式，催化是通过SAM的还原裂解产生甲硫氨酸和5 ′-脱氧腺苷自由基来启动的。氢原子从脱硫生物素转移到该自由基导致形成5 ′-脱氧腺苷和脱硫生物素基底物自由基，然后通过与附近[2Fe-2S]2+簇的硫化物形成新的C-S键淬灭。使用第二当量的Na 2 Met的类似反应顺序导致硫杂环的完成。先前证明的形成和衰减的一个可分离的化学中间体，9-巯基脱硫生物素（MDTB），和平行的形成和衰减的一个不寻常的信号，通过电子顺磁共振（EPR）光谱分配给一个[2Fe-2S]+集群观察。本计画将使用同位素标记的脉冲EPR方法及酵素来研究此顺磁性中间体的结构与电子状态。穆斯堡尔谱和质谱将用于确定[2Fe-2S]2+团簇是否在每次转换后再生。此外，定点突变实验将确定[2 Fe-2S]2+簇是否对活性真正至关重要。将通过氘标记和同位素效应分析研究在体外和体内观察到的9-巯基脱硫生物素和生物素形成的异常缓慢的动力学，以确定特定氢转移步骤的限速程度。控制底物活化和C-S键形成的因素的详细描述将有助于所有自由基SAM enzymes.Broader ImpactsThe研究项目描述的机械和结构特征的理解提供了一个很好的论坛，为教学的基本技术参与表征酶反应中间体的本科生和研究生。PI是一个新的本科生生物化学学位课程的组织者和主任，通过该课程，有才华的本科生将通过参加“定向研究”课程参与实验室研究。特别是，夏威夷大学为多样化的学生群体提供服务，其中包括来自科学领域代表性不足的群体的相当大比例的学生。此外，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，被掺入人类营养补充剂中，更重要的是，被掺入动物饲料中。生产生物素（以及其他维生素）的低成本生物方法将有利于社会，有助于降低成本和提高粮食生产效率。

Intellectual Merit: Enzymes can take advantage of the high reactivity of radicals to carry out otherwise difficult chemical transformations. Such enzymes are found in all branches of life, and are particularly ubiquitous in the diverse classes of microbes. A large emerging superfamily of enzymes found in virtually all organisms utilize S-adenosyl-L-methionine (AdoMet or SAM) as a substrate or cofactor to generate substrate or protein radicals. An important subset of the Radical SAM superfamily catalyzes the radical-mediated addition of sulfur to biomolecules. Biotin synthase catalyzes the oxidative addition of sulfur to saturated carbon atoms on dethiobiotin, generating the biotin thiophane ring. Biotin synthase is an iron-sulfur enzyme, and isotopic labeling suggests the sulfur atom incorporated into biotin derives from an iron-sulfur cluster. Our research focuses on understanding the chemical details of radical generation, substrate activation, and sulfur incorporation.In the working hypothesis for the biotin synthase reaction sequence, in a manner identical with all Radical SAM enzymes, catalysis is initiated by reductive cleavage of SAM to generate methionine and a 5´-deoxyadenosyl radical. Hydrogen atom transfer from dethiobiotin to this radical results in formation of 5´-deoxyadenosine and a dethiobiotinyl substrate radical, which is then quenched through formation of a new C-S bond with sulfide from a nearby [2Fe-2S]2+ cluster. A similar reaction sequence using a second equivalent of AdoMet leads to completion of the thiophane ring. There is previously demonstrated formation and decay of an isolable chemical intermediate, 9-mercaptodethiobiotin (MDTB), and the parallel formation and decay of an unusual signal observable by electron paramagnetic resonance (EPR) spectroscopy assigned to a [2Fe-2S]+ cluster. This project will use pulsed EPR methods with isotopically labeled substrates and enzyme to examine the structure and electronic state of this paramagnetic intermediate. Mössbauer spectroscopy and mass spectrometry will be used to determine whether the [2Fe-2S]2+ cluster is regenerated following each turnover. In addition, site-directed mutagenesis experiments will determine whether the [2Fe-2S]2+ cluster is truly essential for activity. The exceptionally slow kinetics observed for the formation of both 9-mercaptodethiobiotin and biotin, both in vitro and in vivo will be investigated by deuterium labeling and isotope effect analysis to determine the extent to which the specific hydrogen trans,fer steps are rate limiting. A detailed description of the factors that control substrate activation and C-S bond formation will contribute to the understanding of mechanistic and structural features common to all Radical SAM enzymes.Broader ImpactsThe research projects described provide an excellent forum for teaching the basic techniques involved in characterizing enzyme reaction intermediates to undergraduate and graduate students. The PI is the organizer and director of a new undergraduate biochemistry degree program, through which talented undergraduate students will become involved in laboratory research through participation in a "Directed Research" course. In particular, the University of Hawaii serves a diverse student population that includes a significant percentage of students from underrepresented groups in science. In addition, an 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 feedstocks. A low-cost biological method for the production of biotin (as well as other vitamins) would benefit society by contributing to less expensive and more efficient food production.