First Biochemical Characterization of a Methylcitric Acid Cycle from Bacillus subtilis Strain 168

Abstract

The genome of Bacillus subtilis strain 168 contains the mother cell metabolic gene (mmg) operon that encodes homologues from the methylcitric acid cycle. We showed that the three genes, mmgDE and yqiQ(mmgF), provide three of the five steps of the methylcitric acid cycle. We also showed that the fourth step can be supplied by citB (aconitase), and we suggest that the fifth missing step, the propionyl-CoA synthetase, is probably skipped because the β-oxidation of methyl-branched fatty acids by the enzymes encoded by mmgABC should produce propionyl-CoA. We also noted interesting enzymology for MmgD and MmgE. First, MmgD is a bifunctional citrate synthase/2-methylcitrate synthase with 2.3-fold higher activity as a 2-methylcitrate synthase. This enzyme catalyzes the formation of either (2S,3R)- or (2R,3S)-2-methylcitrate, but reports of 2-methylcitrate synthases from other species indicated that they produced the (2S,3S) isomer. However, we showed that MmgD and PrpC (from Escherichia coli) in fact produce the same stereoisomer. Second, the MmgE enzyme is not a stereospecific 2-methylcitrate dehydratase because it can dehydrate at least two of the four diastereomers of 2-methylcitrate to yield either (E)-2-methylaconitate or (Z)-2-methylaconitate. We also showed for the first time that the E. coli homologue PrpD exhibited the same lack of stereospecificity. However, the physiological pathways proceed via (Z)-2-methylaconitate, which served as the substrate for the citB enzyme in the synthesis of 2-methylisocitrate. We completed our characterization of this pathway by showing that the 2-methylisocitrate produced by CitB is converted to pyruvate and succinate by the enzyme YqiQ(MmgF).

Bacterial Acid Resistance Toward Organic Weak Acid Revealed by RNA-Seq Transcriptomic Analysis in Acetobacter pasteurianus

“…Originally, 2-methylcitrate cycle is considered as a propionate metabolism pathway, as reported in E. coli (Brock et al, 2002), Salmonella spp. (Horswill and Escalante-Semerena, 2001) and Bacillus subtilis (Reddick et al, 2017). This pathway begins with the formation of propionyl-CoA from propionate conducted by propionyl-CoA synthetase.…”

“…Then, methylisocitrate is cleaved into succinate and pyruvate catalyzed by methylisocitrate lyase. Oxaloacetate is subsequently regained from succinate through TCA cycle, which initiates a new round of 2-methylcitrate cycle (Horswill and Escalante-Semerena, 2001; Reddick et al, 2017; Figure 7). In Salmonella spp., all enzymes of 2-methylcitrate cycle besides TCA cycle are encoded by prpBCDE operon (Horswill and Escalante-Semerena, 2001), which is homologous to operon AS.1610–AS.1614 in A. pasteurianus CGMCC 1.41.…”

Loving the poison: the methylcitrate cycle and bacterial pathogenesis

“…In B. subtilis, lack of an obvious propionyl-CoA synthase and a general inability to grow on propionate suggest that the function of the methylcitric acid cycle in this organism is to cope specifically with propionylCoA, and not propionate, making this pathway potentially distinct from the propionate-metabolizing pathways of other species [42]. What, then, is the source of the propionyl-CoA in these circumstances?…”

“…However, MmgD also has 2-methylcitrate synthase activity, and can likewise condense propionyl-CoA with oxaloacetate to begin the 2-MC cycle. The remaining steps are encoded by mmgEF and citB (aconitase) [42]. Further atypical methylcitrate cycles may be present in organisms that do not grow on propionate as a sole carbon source.…”

2‐Methylcitrate cycle: a well‐regulated controller of Bacillus sporulation

“…To avoid accumulation of propionyl-CoA, both bacteria and fungi have incorporated a strategy to efficiently convert propionyl-CoA into pyruvate via the 2-methylcitrate cycle as a part of an extended tricarboxylic acid (TCA) cycle ( Fig. 1) (Pronk et al, 1994;Textor et al, 1997;Horswill and Escalante-Semerena, 1999a;Reddick et al, 2017). The pathway is initiated by the 2-methylcitrate synthase PrpC to form 2-methylcitrate through condensation of propionyl-CoA with oxaloacetate, followed by an isomerization event via dehydration and rehydration steps controlled by the 2-methylcitrate dehydratase PrpD and aconitase CitB to synthesize 2-methyl-cis-aconitate and 2-methylisocitrate, respectively.…”