The FAD-Dependent Tricarballylate Dehydrogenase (TcuA) Enzyme of Salmonella enterica Converts Tricarballylate into cis -Aconitate

Abstract

ABSTRACT 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 × 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°C. The enzyme was inactivated at 45°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.