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.
Highlights
The extremely halophilic members of the class Halobacteria, known as haloarchaea, are often the predominant habitants in hypersaline environments (Oren, 2002)
The identity of the purified recombinant proteins was confirmed using gel digestion by trypsin followed by LC-mass spectrometry (MS)/MS (Mct: 28 matched peptides with posterior error probability 0; mesaconyl-CoA hydratase (Mch): 32 matched peptides with posterior error probability 0) (Supplementary Tables S4, S5)
Analytical gel filtration indicated a molecular mass of native recombinant enzymes of 71 and 86 kDa for Mct and Mch, TABLE 1 | Purification of recombinant mesaconate CoA-transferase (Mct) and mesaconyl-CoA hydratase (Mch) from H. hispanica
Summary
The extremely halophilic members of the class Halobacteria, known as haloarchaea, are often the predominant habitants in hypersaline environments (Oren, 2002). Other haloarchaea use the recently described methylaspartate cycle for acetate assimilation (Khomyakova et al, 2011; Borjian et al, 2016, 2017). In this cycle, one molecule of acetyl-CoA and oxaloacetate are converted to glutamate via the reactions of the tricarboxylic acid (TCA) cycle and glutamate dehydrogenase. Propionyl-CoA carboxylation leads to methylmalonyl-CoA and subsequently to succinyl-CoA and (through the TCA cycle) to oxaloacetate, closing the cycle Glyoxylate reacts with another acetyl-CoA molecule yielding malate, the final product of the cycle (Figure 1). The functioning of the methylaspartate cycle was experimentally shown in Haloarcula marismortui, Haloarcula hispanica and Natrialba magadii and proposed for many other haloarchaea based on the results of bioinformatic analysis of the distribution of its key enzymes (Khomyakova et al, 2011; Borjian et al, 2016, 2017)
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