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

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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.