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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 ?-methylmalyl-CoA. The cleavage of ?-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.

SUBMITTER: Borjian F

PROVIDER: S-EPMC5592240 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Borjian Farshad F Johnsen Ulrike U Schönheit Peter P Berg Ivan A IA

Frontiers in microbiology 20170906

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 acet ...[more]

PMID: 28932214

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Project description:Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and β-methylmalyl-CoA lyase (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave β-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a β-methylmalyl-CoA lyase reaction in vivo The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

Project description:The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act(TBEA6) (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act(TBEA6) to this protein family. The predicted secondary structure indicates the lack of a C-terminal ?-helix. Act(TBEA6) was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V(max) of 44.6 ?mol min(-1) mg(-1) for succinyl-CoA, which corresponds to a turnover number of 36.0 s(-1) per subunit of Act(TBEA6). For 3SP, the apparent V(max) was determined as 46.8 ?mol min(-1) mg(-1), which corresponds to a turnover number of 37.7 s(-1) per subunit of Act(TBEA6). The apparent K(m) values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus ?act mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acd(TBEA6), located downstream of act(TBEA6).

Dataset Information

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

Publications

Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in <i>Haloarcula hispanica</i>.

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