Propionate oxidation in Escherichia coli : evidence for operation of a methylcitrate cycle in bacteria

Abstract

Escherichia coli grew in a minimal medium on propionate as the sole carbon and energy source. Initially a lag phase of 4-7 days was observed. Cells adapted to propionate still required 1-2 days before growth commenced. Incorporation of (2-13C), (3-13C) or (2H3)propionate into alanine revealed by NMR that propionate was oxidized to pyruvate without randomisation of the carbon skeleton and excluded pathways in which the methyl group was transiently converted to a methylene group. Extracts of propionate-grown cells contained a specific enzyme that catalyses the condensation of propionyl-CoA with oxaloacetate, most probably to methylcitrate. The enzyme was purified and identified as the already-known citrate synthase II. By 2-D gel electrophoresis, the formation of a second propionate-specific enzyme with sequence similarities to isocitrate lyases was detected. The genes of both enzymes were located in a putative operon with high identities (at least 76% on the protein level) with the very recently discovered prp operon from Salmonella typhimurium. The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast. The 13C patterns of aspartate and glutamate are consistent with the further oxidation of pyruvate to acetyl-CoA. Oxaloacetate is predominantly generated via the glyoxylate cycle rather than by carboxylation of phosphoenolpyruvate.

2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans

“…This assumption agrees well with the tentative assignment of the 2-methylisocitrate produced by a Candida lipolytica mutant strain as threo-d S -isomer [15]. The identification of PrpB as 2-methylisocitrate lyase corroborates well with studies of a prpB deletion mutant of Salmonella typhimurium, which contains almost the same prp operon as E. coli [3]. After growth on 20 mm glycerol and 5 mm [2-13 C]propionate, the mutant accumulated small amounts of methylcitrate, 2-methylaconitate and 2-methylisocitrate as identified by 13 C-NMR spectroscopy [16].…”

“…As the prp operon is essential for propionate oxidation, it was assumed that prpB might code for 2-methylisocitrate lyase [3]. This hypothesis was checked by overexpression of prpB in E. coli TOP10 cells using the pQE30 vector, whereby an N-terminal His 6 -tag was attached to the enzyme.…”

Studies of Propionate Toxicity in Salmonella enterica Identify 2-Methylcitrate as a Potent Inhibitor of Cell Growth

“…Enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and E. coli catabolize propionate via the 2-methylcitric acid cycle (23,24). However, when this pathway was blocked in Salmonella, sensitivity to propionate increased dramatically (25), suggesting that intermediates of the 2-methylcitric acid cycle may have a more profound negative effect on cell growth than propionate itself.…”

An Asymmetric Model for Na+-translocating Glutaconyl-CoA Decarboxylases

“…Other Biochemical Techniques-The subunits of the isolated glutaconyl-CoA decarboxylase complex were separated by SDS-PAGE (32), blotted on polyvinylidene difluoride membranes (33), and N-terminally sequenced by Edman degradation (34). Protein concentrations were determined with the Bio-Rad Protein Assay.…”