Present address: Institut für Mikrobiologie der Universität, Herrenhäuser Str. 2, D-30167 Hannover, Germany. These two authors contributed equally to this work.
Oxidation of propionate to pyruvate in Escherichia coli
Involvement of methylcitrate dehydratase and aconitase
Article first published online: 11 DEC 2002
European Journal of Biochemistry
Volume 269, Issue 24, pages 6184–6194, December 2002
How to Cite
Brock, M., Maerker, C., Schütz, A., Völker, U. and Buckel, W. (2002), Oxidation of propionate to pyruvate in Escherichia coli. European Journal of Biochemistry, 269: 6184–6194. doi: 10.1046/j.1432-1033.2002.03336.x
Enzymes: acetyl-CoA synthetase (Acs, EC 18.104.22.168); aconitase B [AcnB, 2-methylisocitrate dehydratase (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate hydro-lyase, EC 22.214.171.124, also 126.96.36.199]; citrate synthase (EC 188.8.131.52); fumarase (EC 184.108.40.206); isocitrate lyase (EC 220.127.116.11); malate dehydrogenase (EC 18.104.22.168); malate synthase (EC 22.214.171.124); methylcitrate dehydratase [(2S,3S)-2-hydroxybutane-1,2,3-tricarboxylate hydro-lyase, PrpD, EC 126.96.36.199]; methylcitrate synthase (EC 188.8.131.52); 2-methylisocitrate lyase (EC 184.108.40.206); phosphoglycerate mutase (EC 220.127.116.11); propanol-preferring alcohol dehydrogenase (EC 18.104.22.168); propionyl-CoA synthetase (EC 22.214.171.124); pyruvate kinase (EC 126.96.36.199); succinate dehydrogenase (EC 188.8.131.52).
- Issue published online: 11 DEC 2002
- Article first published online: 11 DEC 2002
- (Received 28 July 2002, revised 24 October 2002, accepted 28 October 2002)
- methylcitrate dehydratase;
- propionate metabolism;
- prp operon
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 Km of 210 µm for (2R,3S)-2-methylisocitrate but shows no activity with (2S,3S)-methylcitrate. On the other hand, PrpD is specific for (2S,3S)-methylcitrate (Km = 440 µm) 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.