Tricarballylate Catabolism in Salmonella enterica. The TcuB Protein Uses 4Fe-4S Clusters and Heme to Transfer Electrons from FADH2 in the Tricarballylate Dehydrogenase (TcuA) Enzyme
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- 作者：Jeffrey A. Lewis ; Jorge C. Escalante-Semerena
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：August 7, 2007
- 年：2007
- 卷：46
- 期：31
- 页码：9107 - 9115
- 全文大小：272K
- 年卷期：v.46,no.31(August 7, 2007)
- ISSN：1520-4995
文摘
Tricarballylate, a citrate analogue, is considered the causative agent of grass tetany, a ruminantdisease characterized by acute magnesium deficiency. Although the normal rumen flora cannot catabolizetricarballylate, the Gram-negative enterobacterium Salmonella enterica can. An operon dedicated totricarballylate utilization (tcuABC) present in this organism encodes all functions required for tricarballylatecatabolism. Tricarballylate is converted to the cis-aconitate in a single oxidative step catalyzed by theFAD-dependent tricarballylate dehydrogenase (TcuA) enzyme. We hypothesized that the uncharacterizedTcuB protein was required to reoxidize the flavin cofactor in vivo. Here, we report the initial biochemicalcharacterization of TcuB. TcuB is associated with the cell membrane and contains two 4Fe-4S clustersand heme. Site-directed mutagenesis of cysteinyl residues putatively required as ligands of the 4Fe-4Sclusters completely inactivated TcuB function. TcuB greatly increased the Vmax of the TcuA reactionfrom 69 ± 2 to 8200 ± 470 nmol min-1 mg-1; the Km of TcuA for tricarballylate was unaffected. Inhibitionof TcuB activity by an inhibitor of ubiquinone oxidation, 2,5-dibromo-3-methyl-6-isoproylbenzoquinone(DBMIB), implicated the quinone pool as the ultimate acceptor of electrons from FADH2. We propose amodel for the electron flow from FADH2, to the 4Fe-4S clusters, to the heme, and finally to the quinonepool.