In vitro characterization of iron-sulfur cluster enzymes involved in thiamin biosynthesis

Cellular metabolism, the sum of all the biochemical pathways in a cell, is a complex system. All the processes and pathways in the cell are interconnected with each reaction capable of impacting the function of several other reactions.;This thesis describes the results of biochemical studies that were pursued based on genetic results that identified a connection between [Fe-S] cluster metabolism and thiamin biosynthesis. Characterization of a class of conditional thiamin auxotrophs (apbC, apbE, iscA, gshA and yggX ), suggested that two targets, ThiH and ThiC, were affected by the disruption of [Fe-S] metabolism. Work herein confirms that ThiH and ThiC contain a [4Fe-4S] cluster as a cofactor.;The isolation of active ThiC from Salmonella enterica is described. S-Adenosylmethionine (AdoMet) and 5-aminoimidazole ribotide (AIR), in addition to a reduced [4Fe-4S] cluster, are the components necessary for ThiC activity. The isolation and characterization of a backbone free radical on ThiC upon interaction with AdoMet are outlined. Studies addressing the stoichiometry in the ThiC reaction are summarized. All the results described in this thesis work are consistent with the designation of ThiC as a member of the Radical SAM (AdoMet) Superfamily of proteins.;The requirement of specific residues necessary for ThiH activity by mutational analysis is explained. Phenotypic analysis of conditional auxotrophs (ThiH* variants) allowed the conclusion that AdoMet is necessary for ThiH activity and that the residues proposed to hold the [Fe-S] cluster are essential for activity. Isolation of active ThiH from various strain backgrounds (those lacking YggX and GshA, or ApbC) supported the hypothesis that the lack of these enzymes affected thiazole formation by influencing the occupancy of the [Fe-S] cluster of ThiH.;Finally, the role of ThiI as an electron carrier essential for ThiH activity is proposed and supported with initial studies described.;Through this work I determined the physiological relevance of several proteins in the context of the cellular network by extrapolation of a defined biochemical function in a purified in vitro system.