Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica

Abstract

Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C₄-dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica, succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii, and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as characteristic enzymes of this cycle.

Keywords: acetate assimilation; class III CoA-transferases; enoyl-CoA hydratases; haloarchaea; methylaspartate cycle.

FIGURE 1

The methylaspartate cycle as studied in H. hispanica. The characteristic intermediates of the cycle are shown in red. Gm, glutamate mutase; Mal, methylaspartate ammonia lyase; Mct, succinyl-CoA:mesaconate CoA-transferase; Mch, mesaconyl-CoA hydratase; Mcl, β-methylmalyl-CoA lyase; Ms, apparent malate synthase.

FIGURE 2

SDS-PAGE (12.5%) of purification steps of recombinant His-tagged (A) mesaconate CoA-transferase and (B) mesaconyl-CoA hydratase from H. hispanica. M, molecular mass markers; lane 1, cell extract without enzyme activity; lane 2, H. volcanii cell extract with measurable activity of the heterologously produced enzyme; lane 3, Ni-sepharose column; lane 4, purified enzyme after gel filtration column. The target proteins are shown with arrows.

FIGURE 3

Influence of KCl on activities of recombinant (A) mesaconate CoA-transferase and (B) mesaconyl-CoA hydratase from H. hispanica. Activity was measured with 1 mM succinyl-CoA and 10 mM mesaconate (Mct) and with 0.5 mM β-methylmalyl-CoA (Mch).

FIGURE 4

Phylogenetic trees of H. hispanica (A) mesaconate CoA-transferase and (B) mesaconyl-CoA hydratase based on amino acid sequence analysis. Standard NCBI BLASTP searches with mesaconate CoA-transferase (Hah_1336) and mesaconyl-CoA hydratase (Hah_1340) from H. hispanica as the queries were performed, and all sequences with E-value ≤ 1e-100 for Mct and ≤ 1e-20 for Mch were used for the phylogenetic analysis. Tree topology and evolutionary distances were determined by the maximum-likelihood method with Poisson correction. The scale bar represents a difference of 0.1 substitution per site. The branch colors represent the sequences from haloarchaea, red; α-proteobacteria, purple; β-proteobacteria, black; actinobacteria, blue; Firmicutes, green. The groups marked with asterisk (^∗) possess the complete set of the methylaspartate cycle genes, the bacterial Mch sequences with two domains are shown in bold lines. Please note that bacterial one-domain sequences were aligned with the N-terminal domain of haloarchael Mch.

FIGURE 5

Comparison of H. hispanica Mct with other structurally characterized members of class III CoA-transferases. (A) Phylogenetic tree of the structurally characterized class III CoA-transferases. The tree is based on amino acid sequence analysis. Tree topology and evolutionary distances were calculated using the maximum-likelihood method with Poisson correction. The scale bar represents a difference of 0.2 substitutions per site. Numbers at nodes indicate the percentage bootstrap values for the clade of this group in 1,000 replications. List of the species and accession numbers are given in Supplementary Table S3. (B) Pairwise amino acid sequence comparison of mesaconate CoA-transferase from H. hispanica (Mct) and acetyl-CoA:oxalate CoA transferase from E. coli (YfdE). The black rectangle indicates the catalytically important Asp169 residue and the red ones indicate the conserved GNxH loop.