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，在不同种类的微生物中尤为普遍。在几乎所有生物体中都发现了一个大型的酶超家族，它利用s -腺苷- l-蛋氨酸（AdoMet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对生物分子的加成。生物素合成酶催化硫对脱硫生物素上饱和碳原子的氧化加成，生成生物素硫烷环。生物素合成酶是一种铁硫酶，同位素标记表明生物素中的硫原子来自铁硫簇。我们的研究重点是了解自由基生成，底物活化和硫结合的化学细节。在生物素合成酶反应序列的工作假设中，与所有Radical SAM酶一样，催化是通过SAM的还原裂解产生蛋氨酸和5′-脱氧腺苷基自由基。氢原子从脱硫生物素转移到该自由基导致5´-脱氧腺苷和脱硫生物素基底物自由基的形成，然后通过与附近的[2Fe-2S]2+簇中的硫化物形成新的C-S键来淬灭。使用AdoMet的第二等价物进行类似的反应序列，导致硫代环的完成。先前已经证明了一种可分离的化学中间体，9-巯基脱硫生物素（MDTB）的形成和衰变，以及通过电子顺磁共振（EPR）光谱可观察到的一种特殊信号的平行形成和衰变，该信号被分配给[2Fe-2S]+簇。本项目将使用同位素标记底物和酶的脉冲EPR方法来检测这种顺磁中间体的结构和电子状态。Mössbauer光谱和质谱法将用于确定每次翻转后[2Fe-2S]2+簇是否再生。此外，位点定向诱变实验将确定[2Fe-2S]2+簇是否真的对活性至关重要。9-巯基脱硫生物素和生物素在体外和体内形成的异常缓慢的动力学将通过氘标记和同位素效应分析来研究，以确定特定氢转移的程度，之后的步骤是限速的。对控制底物活化和C-S键形成的因素的详细描述将有助于理解所有Radical SAM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和研究生教授酶反应中间体表征所涉及的基本技术提供了一个极好的论坛。PI是一个新的本科生物化学学位项目的组织者和负责人，通过这个项目，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，其中包括相当大比例的来自科学领域代表性不足群体的学生。此外，对生物素合成酶机制的进一步了解将有助于生物工程过量生产生物素的发展。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。生产生物素（以及其他维生素）的低成本生物方法将有助于降低成本和提高粮食生产效率，从而使社会受益。

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.