International Research Fellowship Program: Mechanisms of Sulfur Transfer in Biotin Synthase Catalysis and Iron-Sulfur Cluster Biosynthesis

0301826鲁巴赫国际研究奖学金计划使美国科学家和工程师能够在国外进行三到24个月的研究。该项目的奖项提供了联合研究的机会，并提供了使用国外独特或互补的设施、专业知识和实验条件的机会。这一奖项将支持Jon K.Rubach博士与Marc Fontecave博士在法国格勒诺布尔的约瑟夫·傅立叶大学合作进行为期24个月的研究。该项目的目标是研究大肠杆菌生物素合成酶的催化机制，并确定参与铁-硫簇生物合成的蛋白质和机制。生物素合成酶是一个独特的含铁S簇的酶家族的一部分，该家族利用S-腺苷蛋氨酸作为5‘-脱氧腺苷自由基的来源，该自由基用于促进困难的化学转化。生物素合成酶催化硫转移的机理将通过同位素标记、化学标记和质谱学研究来探讨。这些研究将确定产品生物素中的硫是否通过使用吡哆醛-5‘-磷酸的机制来自半胱氨酸，并将确定生物素合成酶催化过程中半胱氨酸残基的氧化状态。生物素合成酶稳定周转所必需的成分将通过细胞裂解产物的分级来鉴定。铁-S簇代谢改变可能是衰老和神经退行性变的一个因素。铁-S簇生物合成的必要成分和化学机制将通过生物合成途径的体外重组来研究。利用X-射线单晶衍射法对参与铁-S簇合物生物合成的蛋白质和蛋白质络合物的三维结构进行了测定。

0301826RubachThe International Research Fellowship Program enables U.S. scientists and engineers to conduct three to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad. This award will support a twenty-four month research fellowship by Dr. Jon K. Rubach to work with Dr. Marc Fontecave at Universite Joseph Fourier in Grenoble, France.The goals of this project are to examine the mechanisms of catalysis by E. coli biotin synthase and to determine the proteins and mechanisms involved in iron-sulfur cluster biosynthesis. Biotin synthase is part of a unique family of Fe-S cluster-containing enzymes that utilize S-adenosylmethionine as source of 5'-deoxyadenosyl radical, which is used to promote difficult chemical transformations. The mechanism of sulfur transfer catalyzed by biotin synthase will be examined through isotope labeling, chemical labeling and mass spectrometry studies. These studies will determine if the sulfur in the product biotin is donated from cysteine through a mechanism using pyridoxal-5'-phosphate and will determine the oxidation states of the cysteine residues during catalysis by biotin synthase. The components necessary for steady-state turnover of biotin synthase will be identified by fractionation of cell lysate. Altered metabolism of Fe-S clusters could be a factor in aging and neurodegeneration. The necessary components and the chemical mechanisms for Fe-S cluster biosynthesis will be examined through in vitro reconstitution of the biosynthetic pathway. The three dimensional structures of the proteins and protein complexes involved in Fe-S cluster biosynthesis will be determined using x-ray crystallography.