Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway

Abstract

The genus Salmonella is responsible for many illnesses in humans and other vertebrate animals. We report here that Salmonella enterica serovar Typhimurium harbors three transketolases that support the non-oxidative branch of the pentose phosphate pathway. BLAST analysis identified two genes, STM14_2885 and STM14_2886, that together encode a putative transketolase (TktC) with 46-47% similarity to the known TktA and TktB isoforms. Assessing the mRNA and protein expression for each of the three transketolases, we determined that all are expressed in wild-type cells and regulated to varying extent by the alternative sigma factor RpoS. Enzyme assays with lysates from wild-type and transketolase-knockout strains established that TktA is responsible for >88% of the transketolase activity in wild-type cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for V_max (539-1362 μM NADH consumed × sec^-1), K_M (80-739 μM), and catalytic efficiency (1.02 × 10⁸-1.06 × 10⁹ M^-1 × sec^-1) for each substrate tested. The recombinant form of TktC had lower K_M values (23-120 μM), while the V_max (7.8-16 μM NADH consumed × sec^-1) and catalytic efficiency (5.58 × 10⁶-6.07 × 10⁸ M^-1 × sec^-1) were 10- to 100-fold lower. Using a murine model of Salmonella infection, we showed that a strain lacking all three transketolases is avirulent in C57BL/6 mice. These data provide evidence that S. Typhimurium possesses three transketolases that contribute to pathogenesis.