Propionate is the second most abundant organic acid in soil [1]. Consequently, aerobic growing soil microorganisms are supposed to be able to grow at the expense of this carbon source. The main pathways involved in propionate metabolism are that of the methylmalonylCoA pathway and the methylcitrate cycle. The reaction of methylmalonyl-CoA mutase leads to the citric acid cycle intermediate succinyl-CoA but is coenzyme B 12 dependent and therefore unlikely to exist in fungi [2].We have shown earlier that the filamentous fungus Aspergillus nidulans metabolizes propionate via the methylcitrate cycle [3][4][5]. The first key enzyme, which is specific for this cycle is the methylcitrate synthase, which catalyses the condensation of propionyl-CoA Methylcitrate synthase is a key enzyme of the methylcitrate cycle and required for fungal propionate degradation. Propionate not only serves as a carbon source, but also acts as a food preservative (E280-283) and possesses a negative effect on polyketide synthesis. To investigate propionate metabolism from the opportunistic human pathogenic fungus Aspergillus fumigatus, methylcitrate synthase was purified to homogeneity and characterized. The purified enzyme displayed both, citrate and methylcitrate synthase activity and showed similar characteristics to the corresponding enzyme from Aspergillus nidulans. The coding region of the A. fumigatus enzyme was identified and a deletion strain was constructed for phenotypic analysis. The deletion resulted in an inability to grow on propionate as the sole carbon source. A strong reduction of growth rate and spore colour formation on media containing both, glucose and propionate was observed, which was coincident with an accumulation of propionyl-CoA. Similarly, the use of valine, isoleucine and methionine as nitrogen sources, which yield propionyl-CoA upon degradation, inhibited growth and polyketide production. These effects are due to a direct inhibition of the pyruvate dehydrogenase complex and blockage of polyketide synthesis by propionylCoA. The surface of conidia was studied by electron scanning microscopy and revealed a correlation between spore colour and ornamentation of the conidial surface. In addition, a methylcitrate synthase deletion led to an attenuation of virulence, when tested in an insect infection model and attenuation was even more pronounced, when whitish conidia from glucose ⁄ propionate medium were applied. Therefore, an impact of methylcitrate synthase in the infection process is discussed.

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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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