Dataset Information

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation.

ABSTRACT: A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO2 fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 micromol min(-1) mg protein(-1). The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA --> 3-hydroxypropionyl-CoA + AMP + PP(i). The enzyme was purified 95-fold to a specific activity of 18 micromol min(-1) mg protein(-1) from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO2 fixation in this archaeon and possibly other members of the Sulfolobaceae family.

SUBMITTER: Alber BE

PROVIDER: S-EPMC2238213 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation.

Alber Birgit E BE Kung Johannes W JW Fuchs Georg G

Journal of bacteriology 20071228 4

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO2 fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 micromol min(-1) mg protein(-1). The reaction sequence is catalyzed by at least two enzymes. The f ...[more]

PMID: 18165310

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Project description:Autotrophic Crenarchaeota use two different cycles for carbon dioxide fixation. Members of the Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle, whereas Desulfurococcales and Thermoproteales use the dicarboxylate/4-hydroxybutyrate cycle. While these two cycles differ in the carboxylation reactions resulting in the conversion of acetyl-CoA + 2 CO2 to succinyl-CoA, they have a common regeneration part in which succinyl-CoA is reconverted to two acetyl-CoA molecules. This common part includes crotonyl-CoA conversion to acetoacetyl-CoA, which has unequivocally been shown in Ignicoccus hospitalis (Desulfurococcales) and Pyrobaculum neutrophilus (Thermoproteales) to be catalyzed by a bifunctional crotonase/3-hydroxybutyryl-CoA dehydrogenase. It is a fusion protein consisting of an enoyl-CoA hydratase and a dehydrogenase domain. As the homologous bifunctional protein is present in Sulfolobales as well, its common functioning in the conversion of crotonyl-CoA to acetoacetyl-CoA was proposed. Here we show that a model autotrophic member of Sulfolobales, Metallosphaera sedula, possesses in addition to the bifunctional protein (Msed_0399) several separate genes coding for crotonyl-CoA hydratase and (S)-3-hydroxybutyryl-CoA dehydrogenase. Their genes were previously shown to be transcribed under autotrophic and mixotrophic conditions. The dehydrogenase Msed_1423 (and not the bifunctional protein Msed_0399) appears to be the main enzyme catalyzing the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Homologs of this dehydrogenase are the only (S)-3-hydroxybutyryl-CoA dehydrogenases present in all autotrophic Sulfolobales, strengthening this conclusion. Two uncharacterized crotonase homologs present in M. sedula genome (Msed_0336 and Msed_0384) were heterologously produced and characterized. Both proteins were highly efficient crotonyl-CoA hydratases and may contribute (or be responsible) for the corresponding reaction in the HP/HB cycle in vivo.

Project description:The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA ?-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.

Project description:Since the discovery of symbioses between sulfur-oxidizing (thiotrophic) bacteria and invertebrates at hydrothermal vents over 40 years ago, it has been assumed that autotrophic fixation of CO2 by the symbionts drives these nutritional associations. In this study, we investigated "Candidatus Kentron," the clade of symbionts hosted by Kentrophoros, a diverse genus of ciliates which are found in marine coastal sediments around the world. Despite being the main food source for their hosts, Kentron bacteria lack the key canonical genes for any of the known pathways for autotrophic carbon fixation and have a carbon stable isotope fingerprint that is unlike other thiotrophic symbionts from similar habitats. Our genomic and transcriptomic analyses instead found metabolic features consistent with growth on organic carbon, especially organic and amino acids, for which they have abundant uptake transporters. All known thiotrophic symbionts have converged on using reduced sulfur to gain energy lithotrophically, but they are diverse in their carbon sources. Some clades are obligate autotrophs, while many are mixotrophs that can supplement autotrophic carbon fixation with heterotrophic capabilities similar to those in Kentron. Here we show that Kentron bacteria are the only thiotrophic symbionts that appear to be entirely heterotrophic, unlike all other thiotrophic symbionts studied to date, which possess either the Calvin-Benson-Bassham or the reverse tricarboxylic acid cycle for autotrophy.IMPORTANCE Many animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO2, serving as primary producers for their hosts. Here we describe a clade of nonautotrophic sulfur-oxidizing symbionts, "Candidatus Kentron," associated with marine ciliates. They lack genes for known autotrophic pathways and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead, they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely.

Dataset Information

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation.

Publications

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation.

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