title: 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 to electron acceptors in the cell membrane.
abstract: Tricarballylate, a citrate analogue, is considered the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Although the normal rumen flora cannot catabolize tricarballylate, the Gram-negative enterobacterium Salmonella enterica can. An operon dedicated to tricarballylate utilization (tcuABC) present in this organism encodes all functions required for tricarballylate catabolism. Tricarballylate is converted to the cis-aconitate in a single oxidative step catalyzed by the FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme. We hypothesized that the uncharacterized TcuB protein was required to reoxidize the flavin cofactor in vivo. Here, we report the initial biochemical characterization of TcuB. TcuB is associated with the cell membrane and contains two 4Fe-4S clusters and heme. Site-directed mutagenesis of cysteinyl residues putatively required as ligands of the 4Fe-4S clusters completely inactivated TcuB function. TcuB greatly increased the Vmax of the TcuA reaction from 69 +/- 2 to 8200 +/- 470 nmol min-1 mg-1; the Km of TcuA for tricarballylate was unaffected. Inhibition of 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 a model for the electron flow from FADH2, to the 4Fe-4S clusters, to the heme, and finally to the quinone pool.
authors.affiliation: Department of Bacteriology, University of Wisconsin at Madison, 1550 Linden Drive, Madison, Wisconsin 53706, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Lewis
authors.forename: Jorge C
authors.initials: JC
authors.lastname: Escalante-Semerena
mesh_terms: D000156:Aconitic Acid / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004227:Dithionite / Q000737:chemistry / Q000378:metabolism; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D019779:Salmonella enterica / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014233:Tricarboxylic Acids / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014233:Tricarboxylic Acids; D004227:Dithionite; D006418:Heme; D013455:Sulfur; D000156:Aconitic Acid; D010088:Oxidoreductases; C028021:tricarballylic acid