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

0301826 Rubach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二十四个月的研究。 该计划的奖项提供了联合研究的机会，以及使用国外独特或互补的设施，专业知识和实验条件。 该奖项将支持博士24个月的研究奖学金。这个项目的目标是研究大肠杆菌的催化机制。大肠杆菌生物素合成酶，并确定参与铁硫簇生物合成的蛋白质和机制。 生物素合酶是一个独特的含Fe-S簇的酶家族的一部分，其利用S-腺苷甲硫氨酸作为5 '-脱氧腺苷自由基的来源，其用于促进困难的化学转化。 生物素合成酶催化硫转移的机制将通过同位素标记、化学标记和质谱研究来研究。 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 '-磷酸的机制由半胱氨酸提供，并将确定生物素合酶催化过程中半胱氨酸残基的氧化态。 生物素合酶稳态周转所需的组分将通过细胞裂解物的分级分离来鉴定。 Fe-S簇的代谢改变可能是衰老和神经退行性变的一个因素。 通过体外重组的生物合成途径，研究Fe-S簇生物合成的必要组分和化学机制。 将使用X射线晶体学确定参与Fe-S簇生物合成的蛋白质和蛋白质复合物的三维结构。

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.