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1.
J Biol Chem ; 288(9): 6363-70, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23325803

RESUMO

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP(+) and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP(+) and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3'- and 2'-ribose phosphate group of CoA and NADP(+), respectively, but a different one for the common ADP part: the ß-phosphate of CoA aligns with the α-phosphate of NADP(+). Evolution from an NADP(+) to a bispecific NADP(+) and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP(+) binding. Based on the paralogous aspartate-ß-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.


Assuntos
Proteínas Arqueais/química , Coenzima A/química , NADP/química , Oxirredutases/química , Sulfolobus/enzimologia , Sítios de Ligação , Relação Estrutura-Atividade
2.
J Bacteriol ; 194(11): 2894-903, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22447903

RESUMO

The anaerobic metabolism of indoleacetate (indole-3-acetic acid [IAA]) in the denitrifying betaproteobacterium Azoarcus evansii was studied. The strain oxidized IAA completely and grew with a generation time of 10 h. Enzyme activities that transformed IAA were present in the soluble cell fraction of IAA-grown cells but were 10-fold downregulated in cells grown on 2-aminobenzoate or benzoate. The transformation of IAA did not require molecular oxygen but required electron acceptors like NAD(+) or artificial dyes. The first products identified were the enol and keto forms of 2-oxo-IAA. Later, polar products were observed, which could not yet be identified. The first steps likely consist of the anaerobic hydroxylation of the N-heterocyclic pyrrole ring to the enol form of 2-oxo-IAA, which is catalyzed by a molybdenum cofactor-containing dehydrogenase. This step is probably followed by the hydrolytic ring opening of the keto form, which is catalyzed by a hydantoinase-like enzyme. A comparison of the proteome of IAA- and benzoate-grown cells identified IAA-induced proteins. Owing to the high similarity of A. evansii with strain EbN1, whose genome is known, we identified a cluster of 14 genes that code for IAA-induced proteins involved in the early steps of IAA metabolism. These genes include a molybdenum cofactor-dependent dehydrogenase of the xanthine oxidase/aldehyde dehydrogenase family, a hydantoinase, a coenzyme A (CoA) ligase, a CoA transferase, a coenzyme B(12)-dependent mutase, an acyl-CoA dehydrogenase, a fusion protein of an enoyl-CoA hydratase and a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, and a periplasmic substrate binding protein for ABC transport as well as a transcriptional regulator of the GntR family. Five predicted enzymes form or act on CoA thioesters, indicating that soon after the initial oxidation of IAA and possibly ring opening, CoA thioesters are formed, and the carbon skeleton is rearranged, followed by a CoA-dependent thiolytic release of another CoA thioester. We propose a scheme of an anaerobic IAA metabolic pathway that ultimately leads to 2-aminobenzoyl-CoA or benzoyl-CoA.


Assuntos
Azoarcus/metabolismo , Ácidos Indolacéticos/metabolismo , Anaerobiose , Azoarcus/enzimologia , Azoarcus/genética , Azoarcus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Redes e Vias Metabólicas , Dados de Sequência Molecular
3.
J Bacteriol ; 193(5): 1201-11, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21169482

RESUMO

Two autotrophic carbon fixation cycles have been identified in Crenarchaeota. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the Thermoproteales and Desulfurococcales. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic Sulfolobales; a similar cycle may operate in autotrophic aerobic marine Crenarchaeota. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of succinyl-CoA to two molecules of acetyl-CoA in Thermoproteus neutrophilus (Thermoproteales), Ignicoccus hospitalis (Desulfurococcales), and Metallosphaera sedula (Sulfolobales). The identified enzymes/genes include succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.


Assuntos
Ciclo do Carbono/genética , Ciclo do Carbono/fisiologia , Crenarchaeota/enzimologia , Crenarchaeota/genética , Regulação da Expressão Gênica em Archaea/fisiologia , Acetil-CoA C-Aciltransferase , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Processos Autotróficos/fisiologia , Perfilação da Expressão Gênica , Hidroxibutirato Desidrogenase/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
4.
J Bacteriol ; 193(5): 1191-200, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21169486

RESUMO

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹4C]butyrate and [1,4-¹³C1]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C4intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.


Assuntos
Proteínas de Bactérias/metabolismo , Ciclo do Carbono/fisiologia , Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Sulfolobaceae/enzimologia , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Isótopos de Carbono , Metabolismo Energético , Sulfolobaceae/metabolismo
5.
Proc Natl Acad Sci U S A ; 105(22): 7851-6, 2008 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-18511565

RESUMO

Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO(2) fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to pyruvate. Pyruvate is converted to phosphoenol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO(2) acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, beta-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-(14)C]4-hydroxybutyrate, [1,4-(13)C(2)], [U-(13)C(4)]succinate, or [1-(13)C]pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO(2) fixation mechanism.


Assuntos
Acetilcoenzima A/biossíntese , Dióxido de Carbono/metabolismo , Desulfurococcaceae/metabolismo , Ácidos Dicarboxílicos/metabolismo , Hidroxibutiratos/metabolismo , Aminoácidos/metabolismo , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Proteínas/metabolismo , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismo
6.
J Bacteriol ; 191(20): 6352-62, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19684143

RESUMO

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO(2) 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.


Assuntos
Acil Coenzima A/metabolismo , Proteínas Arqueais/metabolismo , Malondialdeído/análogos & derivados , Oxirredutases/metabolismo , Sulfolobales/enzimologia , Ácido gama-Aminobutírico/análogos & derivados , Processos Autotróficos , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Malondialdeído/metabolismo , Proteínas Recombinantes , Sulfolobales/metabolismo , Ácido gama-Aminobutírico/metabolismo
7.
J Bacteriol ; 191(14): 4572-81, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19429610

RESUMO

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO(2) 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.


Assuntos
Acil Coenzima A/metabolismo , Proteínas Arqueais/metabolismo , Enoil-CoA Hidratase/metabolismo , Hidroxibutiratos/metabolismo , Oxirredutases/metabolismo , Propionatos/metabolismo , Sulfolobales/enzimologia , Proteínas Arqueais/isolamento & purificação , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/isolamento & purificação , Genes Arqueais , Redes e Vias Metabólicas , Modelos Biológicos , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Homologia de Sequência de Aminoácidos , Sulfolobales/genética
8.
Nat Rev Microbiol ; 8(6): 447-60, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20453874

RESUMO

The acquisition of cellular carbon from inorganic carbon is a prerequisite for life and marked the transition from the inorganic to the organic world. Recent theories of the origins of life assume that chemo-evolution took place in a hot volcanic flow setting through a transition metal-catalysed, autocatalytic carbon fixation cycle. Many archaea live in volcanic habitats under such constraints, in high temperatures with only inorganic substances and often under anoxic conditions. In this Review, we describe the diverse carbon fixation mechanisms that are found in archaea. These reactions differ fundamentally from those of the well-known Calvin cycle, and their distribution mirrors the phylogenetic positions of the archaeal lineages and the needs of the ecological niches that they occupy.


Assuntos
Archaea/metabolismo , Processos Autotróficos , Carbono/metabolismo , Acetilcoenzima A/metabolismo , Archaea/classificação , Transportadores de Ácidos Dicarboxílicos/metabolismo , Ecossistema , Glucose/metabolismo , Hidroxibutiratos/metabolismo , Redes e Vias Metabólicas , Filogenia
9.
Science ; 318(5857): 1782-6, 2007 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-18079405

RESUMO

The assimilation of carbon dioxide (CO2) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling.


Assuntos
Dióxido de Carbono/metabolismo , Hidroxibutiratos/metabolismo , Ácido Láctico/análogos & derivados , Sulfolobaceae/metabolismo , Acetilcoenzima A/metabolismo , Acetil-CoA Carboxilase/metabolismo , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Anaerobiose , Archaea/genética , Archaea/metabolismo , Processos Autotróficos , Bicarbonatos/metabolismo , Genes Arqueais , Hidroliases/genética , Hidroliases/metabolismo , Cinética , Ácido Láctico/metabolismo , Redes e Vias Metabólicas , Dados de Sequência Molecular , Oxirredução , Fotossíntese , Filogenia , Sulfolobaceae/genética
10.
J Bacteriol ; 188(24): 8551-9, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17041055

RESUMO

Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --> malonate-semialdehyde + CoA + NADP+. In its native state it is associated with small RNA. Its activity was stimulated by Mg2+ and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.


Assuntos
Ácido Láctico/análogos & derivados , Oxirredutases/metabolismo , Sulfolobaceae/enzimologia , Sulfolobus/enzimologia , Sequência de Aminoácidos , Dióxido de Carbono/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Láctico/metabolismo , Dados de Sequência Molecular , NADP/metabolismo , Oxirredutases/química , Oxirredutases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sulfolobaceae/genética , Sulfolobaceae/crescimento & desenvolvimento , Sulfolobus/genética , Sulfolobus/crescimento & desenvolvimento
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