Abstract
The 2-methylcitric acid cycle (2-MCC) is a common route of propionate catabolism in microorganisms. In Salmonella enterica, the prpBCDE operon encodes most of the 2-MCC enzymes. In other organisms, e.g., Shewanella oneidensis MR-1, two genes, acnD and prpF replace prpD, which encodes 2-methylcitrate dehydratase. We showed that together, S. oneidensis AcnD and PrpF (SoAcnD, SoPrpF) compensated for the absence of PrpD in a S. enterica prpD strain. We also showed that SoAcnD had 2-methylcitrate dehydratase activity and that PrpF has aconitate isomerase activity. Here we report in vitro evidence that the product of the SoAcnD reaction is an isomer of 2-methyl-cis-aconitate (2-MCA], the product of the SePrpD reaction. We show that the SoPrpF protein isomerizes the product of the AcnD reaction into the PrpD product (2-MCA], a known substrate of the housekeeping aconitase (AcnB]. Given that SoPrpF is an isomerase, that SoAcnD is a dehydratase, and the results from in vivo and in vitro experiments reported here, it is likely that 4-methylaconitate is the product of the AcnD enzyme. Results from in vivo studies using a S. enterica prpD strain show that SoPrpF variants with substitutions of residues K73 or C107 failed to support growth with propionate as the sole source of carbon and energy. High-resolution (1.22 Å) three-dimensional crystal structures of PrpFK73E in complex with trans-aconitate or malonate provide insights into the mechanism of catalysis of the wild-type protein.
Highlights
The 2-methylcitric acid cycle (2-MCC) (Fig 1) is widely distributed route of propionate catabolism in microorganisms
After a 1.5-hr incubation period, the reactions mixtures were acidified with H2SO4 to a final concentration of 5 mM, and a 100 μl sample of the reaction was resolved by High-performance liquid chromatography (HPLC)
We show that the PrpF protein of S. oneidensis (SoPrpF) has isomerase activity that converts the product of the SoAcnD reaction into 2-methyl-cis-aconitate, which can be converted into 2-methylisocitrate by aconitase
Summary
The 2-methylcitric acid cycle (2-MCC) (Fig 1) is widely distributed route of propionate catabolism in microorganisms. PrpB encodes 2-methylisocitrate lyase (EC 4.2.1.99) [2, 13, 14]; prpC encodes the 2-methylcitrate synthase (EC 2.3.3.5) (2); prpD, encodes the 2-methylcitrate dehydratase (EC 4.2.1.79) [15]; and prpE encodes the propionyl-CoA synthetase (EC 6.2.1.17) [16, 17]. Some prp operons lack the 2-methylcitrate dehydratase prpD, but rather contain two other genes, acnD and prpF [15]. In S. oneidensis the sequence of prp genes is prpR prpB prpC acnD prpF compared to the S. enterica sequence prpR prpB prpC prpD prpE. In S. oneidensis, the prpE gene encoding propionyl-CoA synthetase is >1.5 Mbp away from the prp operon. AcnD and PrpF activities are necessary and sufficient to compensate for the lack of PrpD during growth with propionate of a prpD strain of S. enterica [18]
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