TheacnDGenes ofShewenella oneidensisandVibrio choleraeEncode a New Fe/S-Dependent 2-Methylcitrate Dehydratase Enzyme That RequiresprpFFunction In Vivo

Abstract

The propionate utilization operons of several bacteria differ from each other in the occurrence of two genes, acnD and prpF, in place of or in addition to the prpD gene encoding an Fe/S-independent 2-methylcitrate dehydratase enzyme. We cloned the acnD and prpF genes from two organisms, Shewanella oneidensis and Vibrio cholerae, and found that, together, the AcnD and PrpF proteins restored the ability of a prpD mutant strain of Salmonella enterica to grow on propionate as a source of carbon and energy. However, neither acnD nor prpF alone was able to substitute for prpD. The AcnD and PrpF proteins were isolated and biochemically analyzed. The AcnD protein required reconstitution of an Fe/S cluster for activity. All detectable AcnD activity was lost after incubation with iron-chelating agents, and no AcnD activity was observed after attempted reconstitution without iron. Nuclear magnetic resonance spectroscopy and in vitro activity assay data showed that AcnD dehydrated 2-methylcitrate and citrate to 2-methyl-cis-aconitate and cis-aconitate, respectively; AcnD also hydrated cis-aconitate. However, 2-methylisocitrate and isocitrate were not substrates for AcnD, indicating that AcnD only catalyzes the first half of the aconitase-like dehydration reactions. No aconitase-like activity was found for PrpF. It is hypothesized that, in vivo, PrpF is an accessory protein required to prevent oxidative damage of the Fe/S center of active AcnD enzyme or that it may be involved in synthesis or repair of the Fe/S cluster present in AcnD.

Genomic Analysis of Carbon Source Metabolism of Shewanella oneidensis MR-1: Predictions versus Experiments

“…Propionate could be metabolized by a variation on the 2-methyl citrate pathway in which 2-methyl citrate is converted to 2-methyl-cis-aconitate by the AcnD-PrpF combination, a complex 2-methyl citrate dehydratase. In this respect, S. oneidensis MR-1 is like P. aeruginosa and Vibrio cholerae, not like E. coli, which has the PrpD version of the enzyme (8).…”

Loving the poison: the methylcitrate cycle and bacterial pathogenesis

“…1), an alternative route is to convert 2-methylcitrate into 4MCA (4-methyl-cis-aconitate, in a reaction catalyzed by AcnD) first and then isomerize the 4MCA into 2MCA (in a reaction catalyzed by PrpF) [9]. Quite why these different strategies for converting 2MC into 2MCA are employed is not clear, although one possibility is that since AcnD is an oxygen-sensitive Fe-S protein, PrpD functions when oxygen levels are high [10]. Some organisms, such as Neisseria meningitides, have also been shown to encode additional genes outside of this 'extended set', presumably providing even greater flexibility in propionate metabolism [11].…”

Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase

“…ICL (PA2634), encoded by aceA, is specific to the glyoxylate shunt pathway and allows a variety of organisms to grow on fatty acids or acetate as a sole carbon source. PA0794 encodes an uncharacterized aconitate hydratase that may substitute for AcnA or AcnB in the tricarboxylic acid (TCA) pathway or may function in propionate metabolism as suggested by Grimek & Escalante-Semerena (2004). L-Asparaginase I (PA2253) converts asparagine into aspartate, which is one step away from conversion to oxaloacetate.…”