TAILIEUCHUNG - Báo cáo Y học: Oxidation of propionate to pyruvate in Escherichia coli Involvement of methylcitrate dehydratase and aconitase

The pathway of the oxidation of propionate to pyruvate in Escherichia coliinvolves 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-methyl-isocitrate requires anovel enzyme,methylcitratedehydratase (PrpD), and the well-known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle. | Eur. J. Biochem. 269 6184-6194 2002 FEBS 2002 doi Oxidation of propionate to pyruvate in Escherichia coli Involvement of methylcitrate dehydratase and aconitase Matthias Brock1 Claudia Maerker1 Alexandra Schutz1 Uwe Volker1 2 t and Wolfaana Buckel1 1 Laboratorium fur Mikrobiologie Fachbereich Biologie Philipps-Universitat Marburg Germany 2Abteilung Biochemie Max-Planck-Institut fur terrestrische Mikrobiologie Marburg Germany 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-methyl-isocitrate requires a novel enzyme methylcitrate dehydratase PrpD and the well-known enzyme aconitase AcnB of the tricarboxylic acid cycle. AcnB was purified as 2-methyl-aconitate hydratase from E. coli cells grown on propionate and identified by its N-terminus. The enzyme has an apparent Km of 210 M 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 M 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 .

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