The FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme of Salmonella enterica converts tricarballylate into cis-aconitate

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 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.

FIG. 1.

Model for tricarballylate catabolism in S. enterica. TcuA oxidizes tricarballylate to cis-aconitate, which can then be catabolized via the Krebs cycle. We propose TcuB is needed to reoxidize FADH₂ bound to TcuA to allow further rounds of catalysis. TCA, tricarboxylic acid.

FIG. 2.

Absorption spectrum for TcuA. The absorbance peaks at 377 and 455 nm are diagnostic of a flavoprotein. The inset shows the absorption spectra for authentic FAD (top) and FMN (bottom).

FIG. 3.

HPLC and ESIMS analyses of the flavin from TcuA. Chromatograms of either authentic FAD monitored at 264 nm (A) or the flavin isolated from heat-denatured TcuA (B) are shown. Numbers represent time (in minutes) of elution after injection. The fractions containing either authentic FAD (C) or the flavin isolated from TcuA (D) had identical mass spectra. The signal with an m/z of 784.8 was consistent with the negative ion of FAD (786 Da).

FIG. 4.

Oligomeric state of TcuA. Known protein standards (open circles) were used to calculate a standard curve. TcuA (solid circle) eluted with an apparent molecular mass of 48 kDa. The r² value for the linear regression of the standard curve was 0.93.

FIG. 5.

HPLC and ESIMS analyses of the TcuA reaction product. Chromatograms of components of the reaction mixture monitored at 210 nm either with heat-inactivated TcuA (A) or with active TcuA (B) are shown. Panel C represents a sample of the TcuA reaction spiked with 50 μM authentic cis-aconitate. Numbers represent time (in minutes) of elution after injection. Tcb denotes tricarballylate, and CA denotes cis-aconitate. Panel D represents the ESIMS analysis of authentic tricarballylate. The signal with an m/z of 175.4 represents the negative ion of tricarballylic acid (176 Da). Panel E represents the ESIMS analysis of the TcuA reaction product. The signal with an m/z of 173.4 was consistent with the negative ion of cis-aconitate. The signal with an an m/z of 175.4 is presumed to be unconsumed tricarballylate.

FIG. 6.

pH and temperature profile for TcuA. Panel A shows the specific activity of TcuA over various pH ranges. Error measurements are presented as standard deviation. Panel B shows product accumulation over time at various temperatures: 25°C; closed squares; 30°C; closed diamonds; 37°C; open squares; 45°C, no detectable activity. Error bars denote standard deviation.

FIG. 7.

Kinetic parameters of TcuA. Initial velocities were calculated by measuring product formation after a 60-min reaction. The error bars denote standard deviation. The inset shows a Lineweaver-Burk plot of the data.