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 language | English (US) |
|---|---|
| Pages (from-to) | 11271-11282 |
| Number of pages | 12 |
| Journal | Journal of Biological Chemistry |
| Volume | 293 |
| Issue number | 29 |
| DOIs | |
| State | Published - Jan 1 2018 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
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 journal › Article
}
TY - JOUR
T1 - Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway
AU - Shaw, Jeff A.
AU - Henard, Calvin A.
AU - Liu, Lin
AU - Dieckman, Lynne
AU - Vázquez-Torres, Andrés
AU - Bourret, Travis J.
PY - 2018/1/1
Y1 - 2018/1/1
N2 - 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.
AB - 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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U2 - 10.1074/jbc.RA118.003661
DO - 10.1074/jbc.RA118.003661
M3 - Article
VL - 293
SP - 11271
EP - 11282
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 29
ER -