Characterization of the Central Metabolic Pathways in
            <i>Thermoanaerobacter</i>
            sp. Strain X514 via Isotopomer-Assisted Metabolite Analysis

Feng, Xueyang; Mouttaki, Housna; Lin, Liwu; Huang, Rick; Wu, Bing; Hemme, Christopher L.; He, Zhili; Zhang, Baichen; Hicks, Leslie M.; Xu, Jian; Zhou, Jizhong; Tang, Yinjie J.

doi:10.1128/aem.00715-09

Cited by 57 publications

(60 citation statements)

References 33 publications

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“…From their detailed analysis, the authors concluded that a Re-type citrate synthase (Re-CS) (EC 2.3.3.3) was present. Similar studies were recently done for several other strictly anaerobic bacteria (3,5,8,32). Such atypical citrate synthases of the Re type were first observed in the strictly anaerobic firmicute Clostridium kluyveri (11) but were later also found in other strictly anaerobic bacteria (9,10,21).…”

supporting

confidence: 59%

“…The resulting labeled [ 13 C]citrate was subsequently cleaved to oxaloacetate plus acetate with Si-citrate lyase in the presence of NADH and malate dehydrogenase so that the oxaloacetate formed was converted to malate. Therefore, the expected products formed from Si-citrate synthase were malate plus [2][3][4][5][6][7][8][9][10][11][12][13] C]acetate, whereas in the case of Re-citrate synthase the products were [2][3][4][5][6][7][8][9][10][11][12][13] C]malate plus acetate (21). The stereospecificity of the purified enzyme was corroborated by detecting 13 C-labeled and unlabeled malic acid produced during the citrate cleavage both in the Si-citrate synthase and in the recombinant enzyme assays.…”

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confidence: 72%

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Identification and Characterization of a Re -Citrate Synthase in Dehalococcoides Strain CBDB1

et al. 2011

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The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn 2؉ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-13 C]acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and 13 C-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.Dehalococcoides species are known for their ability to use a wide range of persistent halogenated compounds as terminal electron acceptors in an anaerobic respiration with hydrogen as the electron donor. Apart from this organohalide respiration, no other mode of energy conservation has been described for Dehalococcoides species, although several strains have been isolated and physiologically characterized (1,4,12,13,23). Also, genome analyses of several Dehalococcoides strains have not indicated further energy fixation capabilities (20,24,29).The anabolism of Dehalococcoides species relies on acetate as a carbon source (1, 23). In our model organism, Dehalococcoides strain CBDB1, six acyl coenzyme A (acyl-CoA) (EC 6.2

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confidence: 59%

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confidence: 72%

Identification and Characterization of a Re -Citrate Synthase in Dehalococcoides Strain CBDB1

et al. 2011

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“…Glutamate derived from [1-13 C]acetate was mainly labeled on C-1 (␣-carboxyl group) in Dehalococcoides strains (19,51,52), and this labeling pattern was consistent with that of glutamate in Thermoanaerobacter sp. (16) and Desulfovibrio vulgaris (18). Especially, the pattern of glutamate (C-1 and C-3) in Dehalococcides strains is exactly in accord with the predicted pattern (Fig.…”

Section: Discussionsupporting

confidence: 72%

“…Moreover, there are an increasing number of anaerobic bacteria containing Re-citrate synthase (16)(17)(18)(19)(20). As shown in Fig.…”

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confidence: 88%

Identification and Characterization ofRe-Citrate Synthase in Syntrophus aciditrophicus

Kim

McInerney

2013

J Bacteriol

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“…The dynamic isotope labeling approach (kinetic flux profiling) that we use here is different from the steady-state isotopic approach (metabolic flux analysis) recently used in similar contexts (6,29). One major advantage of our approach is that it provides absolute fluxes throughout the network instead of just ratios of fluxes at branch points.…”

Section: Discussionmentioning

confidence: 99%

Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

et al. 2010

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Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/␣-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.In soil ecology, obligatory anaerobic bacteria are key contributors to the putrefaction of dead organic matter (18). In the human intestine, they are the dominant flora, playing a central role in metabolism, immunity, and disease (16,24,30). Obligatory anaerobes also encompass some of the most promising bioenergy organisms. The soil bacterium Clostridium acetobutylicum is capable of fermenting carbohydrates into hydrogen gas and solvents (acetone, butanol, and ethanol). During World War I, it was used to develop an industrial starch-based process for the production of acetone and butanol that remained the major production route for these solvents during the first half of the last century (5). Since then, and particularly during the last few decades, an active research area has developed to understand and manipulate the metabolism of this organism with the goal of improving hydrogen and solvent production (5, 15). Despite this long history, there are still key pathways of primary metabolism in C. acetobutylicum that remain unresolved. In particular, as is common for most anaerobic bacteria, the tricarboxylic acid (TCA) cycle remains illdefined (14,23,28).C. acetobutylicum is capable of growing on minimal medium (i.e., using glucose as the sole carbon source) (20), and it therefore must be able to synthesize ␣-ketoglutarate, the carbon skeleton of the glutamate family of amino acids. Its genome sequence, however, lacks obvious homologues of many of the enzymes of the TCA cycle, including citrate synthase, ␣-ketoglutarate dehydrogenase, succinyl-coenzyme A (CoA) synthetase, and fumarate reductase/succinate dehydrogenase (23). The apparent lac...

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Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp. Strain X514 via Isotopomer-Assisted Metabolite Analysis

Cited by 57 publications

References 33 publications

Identification and Characterization of a Re -Citrate Synthase in Dehalococcoides Strain CBDB1

Identification and Characterization of a Re -Citrate Synthase in Dehalococcoides Strain CBDB1

Identification and Characterization ofRe-Citrate Synthase in Syntrophus aciditrophicus

Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

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