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

Jeff A. Shaw, Calvin A. Henard, Lin Liu, Lynne Dieckman, Andrés Vázquez-Torres, Travis J. Bourret

Research output: Contribution to journalArticle

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 WT cells and regulated to varying extents by the alternative sigma factor RpoS. Enzyme assays with lysates from WT and transketolase-knockout strains established that TktA is responsible for >88% of the transketolase activity in WT cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for Vmax (539 –1362 M NADH consumed/s), Km (80 –739 M), and catalytic efficiency (1.02 108-1.06 109 M1/s) for each substrate tested. The recombinant form of TktC had lower Km values (23–120 M), whereas the Vmax (7.8 –16 M NADH consumed/s) and catalytic efficiency (5.58 106 to 6.07 108 M1/s) were 10 –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.

Original languageEnglish (US)
Pages (from-to)11271-11282
Number of pages12
JournalJournal of Biological Chemistry
Volume293
Issue number29
DOIs
StatePublished - Jan 1 2018

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Transketolase
Pentoses
Pentose Phosphate Pathway
Salmonella
Salmonella enterica
Phosphates
Enzymes
NAD
Sigma Factor
Salmonella Infections
Enzyme Assays
Ports and harbors
Inbred C57BL Mouse
Isoenzymes
Vertebrates
Assays
Protein Isoforms
Animals
Genes
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway. / Shaw, Jeff A.; Henard, Calvin A.; Liu, Lin; Dieckman, Lynne; Vázquez-Torres, Andrés; Bourret, Travis J.

In: Journal of Biological Chemistry, Vol. 293, No. 29, 01.01.2018, p. 11271-11282.

Research output: Contribution to journalArticle

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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 WT cells and regulated to varying extents by the alternative sigma factor RpoS. Enzyme assays with lysates from WT and transketolase-knockout strains established that TktA is responsible for >88{\%} of the transketolase activity in WT cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for Vmax (539 –1362 M NADH consumed/s), Km (80 –739 M), and catalytic efficiency (1.02 108-1.06 109 M1/s) for each substrate tested. The recombinant form of TktC had lower Km values (23–120 M), whereas the Vmax (7.8 –16 M NADH consumed/s) and catalytic efficiency (5.58 106 to 6.07 108 M1/s) were 10 –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.",
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