The FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme of Salmonella enterica converts tricarballylate into cis-aconitate.
Tricarballylate is the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Tricarballylate toxicity has been attributed to its ability to chelate magnesium and to inhibit aconitase, a Krebs cycle enzyme. Neither the ruminant nor the normal rumen flora can catabolize tricarballylate to ameliorate its toxic effects. However, the gram-negative enterobacterium Salmonella enterica can use tricarballylate as a carbon and energy source, providing an opportunity to study the genes and enzymes required for tricarballylate catabolism. The tricarballylate utilization (tcu) genes are organized into two transcriptional units, i.e., tcuR and tcuABC. Here, we report the initial biochemical analysis of TcuA. TcuA catalyzed the oxidation of tricarballylate to cis-aconitate. The apparent K(m) of TcuA for tricarballylate was 3.8 +/- 0.4 mM, with a V(max) of 7.9 +/- 0.3 mM min(-1), turnover number (k(cat)) of 6.7 x 10(-2) s(-1), and a catalytic efficiency (k(cat)/K(m)) of 17.8 M(-1) s(-1). Optimal activity was measured at pH 7.5 and 30 degrees C. The enzyme was inactivated at 45 degrees C. One mole of FAD was present per mole of TcuA. We propose a role for TcuB as an electron shuttle protein responsible for oxidizing FADH(2) back to FAD in TcuA.
Lewis
Jeffrey A
JA
Department of Bacteriology, University of Wisconsin-Madison, 1710 University Avenue, Madison, WI 53726-4087, USA.
Escalante-Semerena
Jorge C
JC
D000156:Aconitic Acid / Q000378:metabolism; D019343:Citric Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D019779:Salmonella enterica / Q000201:enzymology*; D013696:Temperature
D005182:Flavin-Adenine Dinucleotide; D019343:Citric Acid; D000156:Aconitic Acid; D010088:Oxidoreductases