Summary auto-generated
Horswill and Escalante-Semerena identified the prpE gene of Salmonella typhimurium LT2 as encoding propionyl-CoA synthetase, an enzyme essential for propionate catabolism. Using genetic and biochemical approaches, they demonstrated that prpE mutants could still utilize propionate due to compensation by acetyl-CoA synthetase (Acs), but double mutants lacking both PrpE and Acs could not grow on propionate. Cell-free extracts enriched for PrpE catalyzed propionyl-CoA formation in a propionate-, ATP-, Mg2+-, and CoA-dependent manner. The enzyme showed substrate specificity, accepting acetate at 48% the rate of propionate but not butyrate, and requiring ATP exclusively among nucleoside triphosphates. HPLC, mass spectrometry, and UV-visible spectroscopy confirmed propionyl-CoA as the reaction product. Genetic evidence established that prpE is part of the prpBCD operon. This represents the first gene identification for propionyl-CoA synthetase activity, suggesting that similar genes annotated as acetyl-CoA synthetases in other bacteria may actually encode propionyl-CoA synthetases based on sequence homology.
Key findings
- The prpE gene encodes propionyl-CoA synthetase, the first identified gene for this enzymatic activity in bacteria
- Acetyl-CoA synthetase (Acs) can functionally compensate for PrpE deficiency, but both are required for propionate utilization
- PrpE is ATP-dependent and shows substrate specificity for propionate over acetate and butyrate
- The enzyme product was confirmed as propionyl-CoA using HPLC, mass spectrometry, and spectroscopic analysis
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Abstract
Biochemical and genetic evidence is presented to demonstrate that the prpE gene of Salmonella typhimurium encodes propionyl-CoA synthetase, an enzyme required for the catabolism of propionate in this bacterium. While prpE mutants used propionate as carbon and energy source, prpE mutants that lacked acetyl-CoA synthetase (encoded by acs) did not, indicating that Acs can compensate for the lack of PrpE in prpE mutants. Cell-free extracts enriched for PrpE catalysed the formation of propionyl-CoA in a propionate-, ATP-, Mg2+- and HS-CoA dependent manner. Acetate substituted for propionate in the reaction at 48% the rate of propionate; butyrate was not a substrate for PrpE. The propionyl-CoA synthetase activity of PrpE was specific for ATP. GTP, ITP, CTP and TTP were not used as substrates by the enzyme. UV-visible spectrophotometry, HPLC and MS data demonstrated that propionyl-CoA was the product of the reaction catalysed by PrpE.