Oxidation of propionate to pyruvate in Escherichia coli

Abstract

The pathway of the oxidation of propionate to pyruvate in Escherichia coli involves five enzymes, only two of which, methylcitrate synthase and 2-methylisocitrate lyase, have been thoroughly characterized. Here we report that the isomerization of (2S,3S)-methylcitrate to (2R,3S)-2-methylisocitrate requires a novel enzyme, methylcitrate dehydratase (PrpD), and the well-known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle. AcnB was purified as 2-methylaconitate hydratase from E. coli cells grown on propionate and identified by its N-terminus. The enzyme has an apparent K m of 210 lM for (2R,3S)-2-methylisocitrate but shows no activity with (2S,3S)-methylcitrate. On the other hand, PrpD is specific for (2S,3S)-methylcitrate (K m ¼ 440 lM) and catalyses in addition only the hydration of cis-aconitate at a rate that is five times lower. The product of the dehydration of enzymatically synthesized (2S,3S)-methylcitrate was designated cis-2-methylaconitate because of its ability to form a cyclic anhydride at low pH. Hence, PrpD catalyses an unusual syn elimination, whereas the addition of water to cis-2-methylaconitate occurs in the usual anti manner. The different stereochemistries of the elimination and addition of water may be the reason for the requirement for the novel methylcitrate dehydratase (PrpD), the sequence of which seems not to be related to any other enzyme of known function. Northern-blot experiments showed expression of acnB under all conditions tested, whereas the RNA of enzymes of the prp operon (PrpE, a propionyl-CoA synthetase, and PrpD) was exclusively present during growth on propionate. 2D gel electrophoresis showed the production of all proteins encoded by the prp operon during growth on propionate as sole carbon and energy source, except PrpE, which seems to be replaced by acetyl-CoA synthetase. This is in good agreement with investigations on Salmonella enterica LT2, in which disruption of the prpE gene showed no visible phenotype.Keywords: 2-methylisocitrate; aconitase; methylcitrate dehydratase; propionate metabolism; prp operon. Several bacteria and fungi are able to oxidize propionate via methylcitrate to pyruvate. Initially propionyl-CoA condenses with oxaloacetate to (2S,3S)-methylcitrate, which isomerizes to (2R,3S)-2-methylisocitrate. Cleavage leads to pyruvate and succinate. The consecutive oxidative regeneration of oxaloacetate from succinate completes the methylcitrate cycle. Initially this cycle was discovered by growing a mutant strain of the yeast Candida lipolytica on odd-chain fatty acids. The accumulation of a tricarboxylic acid was observed during growth and identified as methylcitrate [1]. Further investigations revealed other enzymes necessary for a functional methylcitrate cycle. The enzymes, however, were only partially characterized and no genomic sequences were identified [2-6]. More recently it was discovered that propionate oxidation in aerobically growing Gram-negative bacteria, especially Escherichia coli [7] and Salmonella enterica serovar Thyphimurium L...

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