The methylaspartate cycle in haloarchaea and its possible role in carbon metabolism

Borjian, Farshad; Han, Jing; Hou, Jing; Xiang, Hua; Berg, Ivan A.

doi:10.1038/ismej.2015.132

Cited by 22 publications

(26 citation statements)

References 59 publications

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“…However, these enzymes participate in haloarchaea in the methylaspartate cycle of acetate assimilation rather than in glutamate fermentation. In this cycle, mesaconate formed in the methylaspartate ammonia-lyase reaction is metabolized through the activation to mesaconyl-CoA rather than via the mesaconase reaction [ 32 , 45 ].…”

Section: Resultsmentioning

confidence: 99%

Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB

Kronen

Berg

2015

PLoS ONE

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Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency k cat/K m, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration.

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Section: Resultsmentioning

confidence: 99%

Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB

Kronen

Berg

2015

PLoS ONE

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“…Mesaconate concentrations were determined as described previously (6). CoA and CoA esters were identified and quantified by UPLC with an RP C 18 column (BEH C 18 , 1.7 m, 2.1 by 100 mm; Waters) as described in reference 16.…”

Section: Methodsmentioning

confidence: 99%

Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica

et al. 2017

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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 ␤-methylmalyl-CoA lyase (haloarchaeal ␤-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 ␤-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 ␤-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, yetto-be-discovered malyl-CoA thioesterase in haloarchaea.IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the highenergy 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.KEYWORDS CitE proteins, acetate assimilation, haloarchaea, thioesterase

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“…Anaplerotic pathways enable growth on acetate, fatty acids and other compounds metabolized through acetyl-CoA, allowing formation of all cellular building blocks from this central metabolite. Over 92% of all sequenced members of haloarchaea possess genes of anaplerotic acetate assimilation pathways ( Borjian et al, 2016 ). About half of haloarchaea use the glyoxylate cycle discovered in Kornberg and Krebs (1957) that functions in all three domains of life ( Serrano et al, 1998 ).…”

Section: Introductionmentioning

confidence: 99%

“…About half of haloarchaea use the glyoxylate cycle discovered in Kornberg and Krebs (1957) that functions in all three domains of life ( Serrano et al, 1998 ). 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.…”

Section: Introductionmentioning

confidence: 99%

“…In sum, the cycle converts two acetyl-CoA molecules into malate. 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 ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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The methylaspartate cycle in haloarchaea and its possible role in carbon metabolism

Cited by 22 publications

References 59 publications

Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB

Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB

Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica

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

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