3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase, Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales

ABSTRACT A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO2 fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.

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Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

Applied and Environmental Microbiology ◽

10.1128/aem.04146-13 ◽

2014 ◽

Vol 80 (8) ◽

pp. 2536-2545 ◽

Cited By ~ 17

Author(s):

Aaron B. Hawkins ◽

Michael W. W. Adams ◽

Robert M. Kelly

Keyword(s):

Carbon Fixation ◽

Coenzyme A ◽

Flux Analysis ◽

Central Metabolism ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Metallosphaera Sedula ◽

Acetyl Coenzyme ◽

Coa Ligase

ABSTRACTThe extremely thermoacidophilic archaeonMetallosphaera 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 CO2molecules 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 β-ketothiolase (Msed_0656), were produced recombinantly inEscherichia coli, combinedin vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2fixation and central metabolism were examined using a gas-intensive bioreactor system in whichM. sedulawas 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 inM. sedulametabolism. 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 CO2fixation and central metabolism inM. sedula.

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Malonic Semialdehyde Reductase, Succinic Semialdehyde Reductase, and Succinyl-Coenzyme A Reductase from Metallosphaera sedula: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Sulfolobales

Journal of Bacteriology ◽

10.1128/jb.00794-09 ◽

2009 ◽

Vol 191 (20) ◽

pp. 6352-6362 ◽

Cited By ~ 50

Author(s):

Daniel Kockelkorn ◽

Georg Fuchs

Keyword(s):

Alcohol Dehydrogenase ◽

Carbon Fixation ◽

Coenzyme A ◽

Succinic Semialdehyde ◽

Metallosphaera Sedula ◽

Semialdehyde Dehydrogenase ◽

Malonyl Coa ◽

Hydroxyacyl Coa Dehydrogenase ◽

Coa Reductase ◽

Coenzyme A Reductase

ABSTRACT A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO2 fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.

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Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

Journal of Bacteriology ◽

10.1128/jb.00752-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8250-8256 ◽

Cited By ~ 73

Author(s):

Keiichi Uchino ◽

Terumi Saito ◽

Birgit Gebauer ◽

Dieter Jendrossek

Keyword(s):

Coenzyme A ◽

Ralstonia Eutropha ◽

Major Product ◽

Acetyl Coa ◽

Native Form ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme ◽

Phb Synthase ◽

Coa Reductase

ABSTRACTPoly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) fromRalstonia eutrophacatalyzed formation of PHB from14C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD+, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD+/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.

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Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

Journal of Bacteriology ◽

10.1128/jb.181.4.1088-1098.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1088-1098 ◽

Cited By ~ 103

Author(s):

Castor Menendez ◽

Zsuzsa Bauer ◽

Harald Huber ◽

Nasser Gad’on ◽

Karl-Otto Stetter ◽

...

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Carbon Fixation ◽

Coenzyme A ◽

Autotrophic Growth ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Carboxylase Activity ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme

ABSTRACT The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed forC. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus andAcidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula,S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

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Investigation of Carbon Metabolism in “Dehalococcoides ethenogenes” Strain 195 by Use of Isotopomer and Transcriptomic Analyses

Journal of Bacteriology ◽

10.1128/jb.00085-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5224-5231 ◽

Cited By ~ 62

Author(s):

Yinjie J. Tang ◽

Shan Yi ◽

Wei-Qin Zhuang ◽

Stephen H. Zinder ◽

Jay D. Keasling ◽

...

Keyword(s):

Genome Annotation ◽

Carbon Fixation ◽

Coenzyme A ◽

Citrate Synthase ◽

Tricarboxylic Acid Cycle ◽

Acetyl Coa ◽

Experimental Conditions ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme ◽

Dehalococcoides Ethenogenes

ABSTRACT Members of the genus “Dehalococcoides” are the only known microorganisms that can completely dechlorinate tetrachloroethene and trichloroethene to the innocuous end product, ethene. This study examines the central metabolism in “Dehalococcoides ethenogenes” strain 195 via 13C-labeled tracer experiments. Supported by the genome annotation and the transcript profile, isotopomer analysis of key metabolites clarifies ambiguities in the genome annotation and identifies an unusual biosynthetic pathway in strain 195. First, the 13C-labeling studies revealed that strain 195 contains complete amino acid biosynthesis pathways, even though current genome annotation suggests that several of these pathways are incomplete. Second, the tricarboxylic acid cycle of strain 195 is confirmed to be branched, and the Wood-Ljungdahl carbon fixation pathway is shown to not be functionally active under our experimental conditions; rather, CO2 is assimilated via two reactions, conversion of acetyl-coenzyme A (acetyl coenzyme A [acetyl-CoA]) to pyruvate catalyzed by pyruvate synthase (DET0724-0727) and pyruvate conversion to oxaloacetate via pyruvate carboxylase (DET0119-0120). Third, the 13C-labeling studies also suggested that isoleucine is synthesized from acetyl-CoA and pyruvate via citramalate synthase (CimA, EC 2.3.1.182), rather than from the common pathway via threonine ammonia-lyase (EC 4.3.1.19). Finally, evidence is presented that strain 195 may contain an undocumented citrate synthase (>95% Re-type stereospecific), i.e., a novel Re-citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri.

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Lack of Citrate Lyase — the Key Enzyme of the Reductive Carboxylic Acid Cycle — in Chlorobium thiosulfatophilum and Rhodospirillum rubrum

Zeitschrift für Naturforschung B ◽

10.1515/znb-1972-0822 ◽

1972 ◽

Vol 27 (8) ◽

pp. 967-973 ◽

Cited By ~ 21

Author(s):

Norbert Beuscher ◽

Gerhard Gottschalk

Keyword(s):

Carboxylic Acid ◽

Rhodospirillum Rubrum ◽

Coenzyme A ◽

Reaction Sequence ◽

Acetyl Coenzyme A ◽

Acid Cycle ◽

Acetyl Coenzyme ◽

Citrate Lyase ◽

Chlorobium Thiosulfatophilum ◽

Key Enzyme

Extracts of Chlorobium thiosulfatophilum and of Rhodospirillum rubrum have been tested for the presence of citrate lyase under various conditions. This enzyme could not be detected. It is, therefore, concluded that a complete reductive carboxylic acid cycle does not occur in these microorganisms.The enzymes required for a-ketoglutarate synthesis from oxaloaetate and acetyl-coenzyme A are present in autotrophically grown cells of C. thiosulfatophilum and R. rubrum. This supports the view that a reaction sequence catalyzing the conversion of α-ketoglutarate into oxaloacetate is unlikely to exist in these bacteria.

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Deconstructing Methanosarcina acetivorans into an acetogenic archaeon

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2113853119 ◽

2022 ◽

Vol 119 (2) ◽

pp. e2113853119

Author(s):

Christian Schöne ◽

Anja Poehlein ◽

Nico Jehmlich ◽

Norman Adlung ◽

Rolf Daniel ◽

...

Keyword(s):

Carbon Monoxide ◽

Energy Conservation ◽

Carbon Fixation ◽

Coenzyme A ◽

Methanogenic Archaea ◽

Methanosarcina Acetivorans ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme ◽

Acetyl Coa Pathway

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide–dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

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Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus, a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO2 Fixation

Journal of Bacteriology ◽

10.1128/jb.184.9.2404-2410.2002 ◽

2002 ◽

Vol 184 (9) ◽

pp. 2404-2410 ◽

Cited By ~ 84

Author(s):

Michael Hügler ◽

Castor Menendez ◽

Hermann Schägger ◽

Georg Fuchs

Keyword(s):

Alcohol Dehydrogenase ◽

Aldehyde Dehydrogenase ◽

Coenzyme A ◽

Specific Activity ◽

Chloroflexus Aurantiacus ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Genome Database ◽

Malonyl Coa ◽

Coa Reductase

ABSTRACT The 3-hydroxypropionate cycle is a new autotrophic CO2 fixation pathway in Chloroflexus aurantiacus and some archaebacteria. The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate. This reduction step was studied in Chloroflexus aurantiacus. A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H+ → malonate semialdehyde + NADP+ + CoA and malonate semialdehyde + NADPH + H+ → 3-hydroxypropionate + NADP+. The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an α2 composition. The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database. Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain. No indication of the presence of a prosthetic group was obtained; Mg2+ and Fe2+ stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent Km values of 30 μM malonyl-CoA and 25 μM NADPH and a turnover number of 25 s−1 subunit−1. The specific activity in autotrophically grown cells was 0.08 μmol of malonyl-CoA reduced min−1 (mg of protein)−1, compared to 0.03 μmol min−1 (mg of protein)−1 in heterotrophically grown cells, indicating downregulation under heterotrophic conditions. Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle. Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides.

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