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[1-14 C]butyrate and [1,4-13 C 1 ]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 C 4 intermediates 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.

SummaryThe enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoylcoenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin-containing 'old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases.

The 2-naphthoyl-coenzyme A (NCoA) reductase (NCR) is so far the only characterized enzyme involved in the anaerobic degradation of the environmentally relevant polycyclic aromatic hydrocarbons. The old yellow enzyme (OYE) family member apparently reduced the nonactivated naphthyl ring to 5,6,7,8-tetrahydro-2-napthoyl-CoA (THNCoA). In this work, the candidate genes of three NCRs from the sulphate-reducing, naphthalene-degrading N47 and NaphS2 cultures were expressed in Escherichia coli. The isolated products contained flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) a [4Fe-4S] cluster and catalyzed only the two-electron reduction of NCoA to 5,6-dihydro-2-naphthoyl-CoA (5,6-DHNCoA) at a very negative E°' = -493 mV. All NCRs exhibited high NCoA-forming DHNCoA oxidase activities that are proposed to be involved in oxygen-detoxification during naphthalene degradation. Extracts of N47 and NaphS2 catalyzed the reduction of 5,6-DHNCoA to THNCoA. Genes putatively coding for 5,6-DHNCR from N47 and NaphS2 were heterologously expressed in E. coli. The enriched enzyme products specifically catalyzed the reduction of 5,6-DHNCoA to THNCoA at E°' = -375 mV. With the three NCRs and two 5,6-DHNCRs, five OYEs have been characterized that are involved in the reduction of the nonsubstituted naphthyl-ring system; these unprecedented enzymatic reactions expand our knowledge of the functional diversity of OYE.

Birch reductions of aromatic hydrocarbons by means of single-electron-transfer steps depend on alkali metals, ammonia, and cryogenic reaction conditions. In contrast, 2-naphthoyl-coenzyme A (2-NCoA) and 5,6-dihydro-2-NCoA (5,6-DHNCoA) reductases catalyze two two-electron reductions of the naphthoyl-ring system to tetrahydronaphthoyl-CoA at ambient temperature. Using a number of substrate analogues, we provide evidence for a Meisenheimer complex-analogous intermediate during 2-NCoA reduction, whereas the subsequent reduction of 5,6-dihydro-2-NCoA is suggested to proceed via an unprecedented cationic transition state. Using vibrational circular dichroism (VCD) spectroscopy, we demonstrate that both enzymatic reductions are highly stereoselective in D O, providing an enantioselective pathway to products inaccessible by Birch reduction. Moreover, we demonstrate the power of VCD spectroscopy to determine the absolute configuration of isotopically engendered alicyclic stereocenters.

Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with 'Archaeoglobus lithotrophicus' being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin-Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in 'A. lithotrophicus', but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO(2)) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-D-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO(2) fixation pathway in 'A. lithotrophicus'.

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The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from β-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop–driven reduction of CO2 by acyl–coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of β-oxidation in (methyl)menaquinone-containing organisms.

Glutaryl-CoA dehydrogenases (GDHs) are FAD containing acyl-CoA dehydrogenases that usually catalyze the dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA with an electron transferring flavoprotein (ETF) acting as natural electron acceptor. In anaerobic bacteria, GDHs play an important role in the benzoyl-CoA degradation pathway of monocyclic aromatic compounds. In the present study, we identified, purified and characterized the benzoate-induced BamOP as the electron accepting ETF of GDH (BamM) from the Fe(III)-respiring Geobacter metallireducens. The BamOP heterodimer contained FAD and AMP as cofactors. In the absence of an artificial electron acceptor, at pH values above 8, the BamMOP-components catalyzed the expected glutaryl-CoA oxidation to crotonyl-CoA and CO 2 ; however, at pH values below 7, the redox-neutral glutaryl-CoA conversion to butyryl-CoA and CO 2 became the dominant reaction. This previously unknown, strictly ETF-dependent coupled glutaryl-CoA oxidation/crotonyl-CoA reduction activity was facilitated by an unexpected two-electron transfer between FAD (BamM) and FAD (BamOP) , as well as by the similar redox potentials of the two FAD cofactors in the substrate-bound state. The strict order of electron/proton transfer and C-C-cleavage events including transient charge-transfer complexes did not allow an energetic coupling of electron transfer and decarboxylation. This explains why it was difficult to release the glutaconyl-CoA intermediate from reduced GDH. Moreover, it provides a kinetic rational for the apparent inability of BamM to catalyze the reverse reductive crotonyl-CoA carboxylation, even under thermodynamically favourable conditions. For this reason reductive crotonyl-CoA carboxylation, a key reaction in C2-assimilation via the ethylmalonyl-CoA pathway, is accomplished by a different crotonyl-CoA carboxylase/reductase via a covalent NADPH/ene-adduct.