benzoyl-CoA Reductase Research Articles

Rodents consuming the same toxic diet harbor a unique functional core microbiome

Gut microbiota are intrinsic to an herbivorous lifestyle, but very little is known about how plant secondary compounds (PSCs), which are often toxic, influence these symbiotic partners. Here we interrogated the possibility of unique functional core microbiomes in populations of two species of woodrat (Neotoma lepida and bryanti) that have independently converged to feed on the same toxic diet (creosote bush; Larrea tridentata) and compared them to populations that do not feed on creosote bush. Leveraging this natural experiment, we collected samples across a large geographic region in the U.S. desert southwest from 20 populations (~ 150 individuals) with differential ingestion of creosote bush and analyzed three gut regions (foregut, cecum, hindgut) using16S sequencing and shotgun metagenomics. In each gut region sampled, we found a distinctive set of microbes in individuals feeding on creosote bush that were more abundant than other ASVs, enriched in creosote feeding woodrats, and occurred more frequently than would be predicted by chance. Creosote core members were from microbial families e.g., Eggerthellaceae, known to metabolize plant secondary compounds and three of the identified core KEGG orthologs (4-hydroxybenzoate decarboxylase, benzoyl-CoA reductase subunit B, and 2-pyrone-4, 6-dicarboxylate lactonase) coded for enzymes that play important roles in metabolism of plant secondary compounds. The results support the hypothesis that the ingestion of creosote bush sculpts the microbiome across all major gut regions to select for functional characteristics associated with the degradation of the PSCs in this unique diet.

Microbial diversity and metabolic pathways linked to benzene degradation in petrochemical-polluted groundwater

The rapid advance in shotgun metagenome sequencing has enabled us to identify uncultivated functional microorganisms in polluted environments. While aerobic petrochemical-degrading pathways have been extensively studied, the anaerobic mechanisms remain less explored. Here, we conducted a study at a petrochemical-polluted groundwater site in Henan Province, Central China. A total of twelve groundwater monitoring wells were installed to collect groundwater samples. Benzene appeared to be the predominant pollutant, detected in 10 out of 12 samples, with concentrations ranging from 1.4 μg/L to 5,280 μg/L. Due to the low aquifer permeability, pollutant migration occurred slowly, resulting in relatively low benzene concentrations downstream within the heavily polluted area. Deep metagenome sequencing revealed Proteobacteria as the dominant phylum, accounting for over 63 % of total abundances. Microbial α-diversity was low in heavily polluted samples, with community compositions substantially differing from those in lightly polluted samples. dmpK encoding the phenol/toluene 2-monooxygenase was detected across all samples, while the dioxygenase bedC1 was not detected, suggesting that aerobic benzene degradation might occur through monooxygenation. Sequence assembly and binning yielded 350 high-quality metagenome-assembled genomes (MAGs), with 30 MAGs harboring functional genes associated with aerobic or anaerobic benzene degradation. About 80 % of MAGs harboring functional genes associated with anaerobic benzene degradation remained taxonomically unclassified at the genus level, suggesting that our current database coverage of anaerobic benzene-degrading microorganisms is very limited. Furthermore, two genes integral to anaerobic benzene metabolism, i.e, benzoyl-CoA reductase (bamB) and glutaryl-CoA dehydrogenase (acd), were not annotated by metagenome functional analyses but were identified within the MAGs, signifying the importance of integrating both contig-based and MAG-based approaches. Together, our efforts of functional annotation and metagenome binning generate a robust blueprint of microbial functional potentials in petrochemical-polluted groundwater, which is crucial for designing proficient bioremediation strategies.

Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams.

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ. For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans, and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments.

The benzoyl-CoA pathway serves as a genomic marker to identify the oxygen requirements in the degradation of aromatic hydrocarbons.

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase (BCL). The anaerobic route is steered by benzoyl-CoA reductase, which promotes benzoyl-CoA breakdown, which is subsequently oxidized. In certain bacteria at low oxygen conditions, the aerobic metabolism of monoaromatic hydrocarbons occurs through the degradation Box pathway. These pathways have undergone experimental scrutiny in Alphaproteobacteria and Betaproteobacteria and have also been explored bioinformatically in representative Betaproteobacteria. However, there is a gap in our knowledge regarding the distribution of the benzoyl-CoA pathway and the evolutionary forces propelling its adaptation beyond that of representative bacteria. To address these questions, we used bioinformatic procedures to identify the BCLs and the lower pathways that transform benzoyl-CoA. These procedures included the identification of conserved motifs. As a result, we identified two motifs exclusive to BCLs, describing some of the catalytic properties of this enzyme. These motifs helped to discern BCLs from other aryl-CoA ligases effectively. The predicted BCLs and the enzymes of lower pathways were used as genomic markers for identifying aerobic, anaerobic, or hybrid catabolism, which we found widely distributed in Betaproteobacteria. Despite these enhancements, our approach failed to distinguish orthologs from a small cluster of paralogs exhibiting all the specified features to predict an ortholog. Nonetheless, the conducted phylogenetic analysis and the properties identified in the genomic context aided in formulating hypotheses about how this redundancy contributes to refining the catabolic strategy employed by these bacteria to degrade the substrates.

Transformation of Methoxylated Aromatic Compounds by Anaerobic Microorganisms

Abstract—Methoxylated aromatic compounds (MAC) are widely distributed in various habitats and are components of lignin, the second most abundant biopolymer on Earth. This review summarizes the results on microbiology, ecology, and biochemistry of anaerobic MAC catabolism in bacteria and archaea. We analyzed the genomes of 46 prokaryotes anaerobically degrading MAC for the presence of O-demethylase, CO-dehydrogenase/acetyl-CoA synthase, and benzoyl-CoA reductase genes, which determine the possibility of methoxydotrophic growth. It was found that facultative anaerobes of the phylum Pseudomonadota do not have any known genetic determinants of anaerobic O-demethylase reaction as well as of aromatic ring reduction. Thus, the MAC transformation by anaerobic microorganisms can be carried out by diverse biochemical mechanisms and probably plays a more significant role in the global carbon cycle than previously supposed.

Complex and flexible catabolism in Aromatoleum aromaticum pCyN1.

Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil-rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant-derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C-H-bond cleavage at the methyl groups of p-cymene/4-ethyltoluene, toluene and p-cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium-wide (one module shared by p-cymene, 4-ethyltoluene, α-phellandrene, α-terpinene, γ-terpinene and limonene) to broad (central benzoyl-CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP-dependent (class I) benzoyl-CoA reductase and four different β-oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low-affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability.

Genome analysis of Thauera chlorobenzoica strain 3CB-1T, a halobenzoate-degrading bacterium isolated from aquatic sediment.

The genus Thauera is characterized by several species and strains with the ability to degrade a variety of aromatic compounds under denitrifying conditions. Thauera chlorobenzoica strain 3CB-1T, isolated from river sediment, has the unique ability to degrade a variety of halobenzoates, such as 3-chlorobenzoate, 3-bromobenzoate, 3-iodobenzoate, and 2-fluorobenzoate, coupled to nitrate reduction. The genome of T. chlorobenzoica strain 3CB-1T has been sequenced, allowing us to gain insights into the molecular basis for the anaerobic degradation of (halo)aromatic compounds. The 3.77-Mb genome contains 3584 genes; 3514 are protein-coding genes of which 198 are likely associated with degradation of aromatic compounds. It has a G + C content of 67.25%. The genome contains two sets of CoA reductase gene clusters, both belonging to class I benzoate-CoA reductases (BCRs). The genes in one of the two clusters differ from the typical BCRs, with low sequence identities, suggesting they might have different substrate specificities. The genome also contains four benzoate-CoA ligase genes. One likely encodes a 3-hydroxybenzoate-CoA ligase, and two others group together with benzoate-CoA ligases from Thauera aromatica. The fourth has a 77% identity to the mbdA gene from Azoarcus sp. CIB, is absent in the T. aromatica genome, and potentially encodes a halobenzoate-CoA ligase. 3-Chlorobenzoate is reductively dechlorinated in T. chlorobenzoica by a benzoyl-CoA reductase.

Tungstoenzymes: Occurrence, Catalytic Diversity and Cofactor Synthesis

Tungsten is the heaviest element used in biological systems. It occurs in the active sites of several bacterial or archaeal enzymes and is ligated to an organic cofactor (metallopterin or metal binding pterin; MPT) which is referred to as tungsten cofactor (Wco). Wco-containing enzymes are found in the dimethyl sulfoxide reductase (DMSOR) and the aldehyde:ferredoxin oxidoreductase (AOR) families of MPT-containing enzymes. Some depend on Wco, such as aldehyde oxidoreductases (AORs), class II benzoyl-CoA reductases (BCRs) and acetylene hydratases (AHs), whereas others may incorporate either Wco or molybdenum cofactor (Moco), such as formate dehydrogenases, formylmethanofuran dehydrogenases or nitrate reductases. The obligately tungsten-dependent enzymes catalyze rather unusual reactions such as ones with extremely low-potential electron transfers (AOR, BCR) or an unusual hydration reaction (AH). In recent years, insights into the structure and function of many tungstoenzymes have been obtained. Though specific and unspecific ABC transporter uptake systems have been described for tungstate and molybdate, only little is known about further discriminative steps in Moco and Wco biosynthesis. In bacteria producing Moco- and Wco-containing enzymes simultaneously, paralogous isoforms of the metal insertase MoeA may be specifically involved in the molybdenum- and tungsten-insertion into MPT, and in targeting Moco or Wco to their respective apo-enzymes. Wco-containing enzymes are of emerging biotechnological interest for a number of applications such as the biocatalytic reduction of CO2, carboxylic acids and aromatic compounds, or the conversion of acetylene to acetaldehyde.

Oxygen detoxification by dienoyl‐CoA oxidase involving flavin/disulfide cofactors

Class I benzoyl-CoA reductases (BCRs) are oxygen-sensitive key enzymes in the degradation of monocyclic aromatic compounds in anaerobic prokaryotes. They catalyze the ATP-dependent reductive dearomatization of their substrate to cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA). An aromatizing 1,5-dienoyl-CoA oxidase (DCO) activity has been proposed to protect BCRs from oxidative damage, however, the gene and its product involved have not been identified, yet. Here, we heterologously produced a DCO from the hyperthermophilic euryarchaeon Ferroglobus placidus that coupled the oxidation of two 1,5-dienoyl-CoA to benzoyl-CoA to the reduction of O2 to water at 80°C. DCO showed similarities to members of the old yellow enzyme family and contained FMN, FAD and an FeS cluster as cofactors. The O2 -dependent activation of inactive, reduced DCO is assigned to a redox thiol switch at Eo ' = -3mV. We propose a catalytic cycle in which the active site FMN/disulfide redox centers are reduced by two 1,5-dienoyl-CoA (reductive half-cycle), followed by two consecutive two-electron transfer steps to molecular oxygen via peroxy- and hydroxyflavin intermediates yielding water (oxidative half-cycle). This work identified the enzyme involved in a unique oxygen detoxification process for an oxygen-sensitive catabolic enzyme.

Tracking functional bacterial biomarkers in response to a gradient of contaminant exposure within a river continuum

Within all aquatic environments, aside from the physical dispersal of dissolved and/or particulate phase contaminants, alteration from both biological and chemical processes are shown to change the chemistry of the parent compounds. Often these alterations can lead to secondary influences because of cooperative microbial processes (i.e. coupled respiratory pathways and/or energy and biodegradation cycles), complicating our understanding of the biological impact that these mobile compounds impose on ecosystem health. The McMurray Formation (MF) (the formation constituting the minable bituminous oil sands) is a natural, ongoing source of hydrocarbon-bound sediments to river ecosystems in the region (via terrestrial and aquatic erosion), providing a natural “mesocosm” to track and characterize the effects of these compounds on regional aquatic primary productivity. Here we characterize the natural, in-situ microbial response to increasing hydrocarbon exposure along a river continuum in the downstream direction. Using the Steepbank River (STB), suspended and bed sediment samples were collected at 3 sites from upstream to downstream, as the water flows into and through the MF. Samples were then analyzed for the active, in-situ gene expression of the microbial communities. Results from both suspended and bed sediments show clear and significant shifts in the microbial metabolic processes within each respective compartment, in response to the elevated polycyclic aromatic compound (PAC) concentrations. Specific genes likely responsible for hydrocarbon breakdown (Alkane Monooxygenase, Benzoyl-CoA Reductase etc.) experience elevated expression levels, while certain energy metabolism genes (nitrogen, sulfur, methane) reveal fundamental shifts in their pathway specificity, indicating an adaptation response in their basic energy metabolism. Expression from suspended sediments reveal subtle yet delayed metabolic response further downstream compared to bed sediments, indicative of the erosion and transport dynamics within a lotic system. These results provide insight into the use of novel clusters of gene biomarkers to track the active, in-situ microbial response of both emerging and legacy contaminants. Such information will be important in determining the best management strategies for the monitoring and assessment of aquatic health in both natural and contaminated ecosystems.

The class II benzoyl-coenzyme A reductase complex from the sulfate-reducing Desulfosarcina cetonica.

Benzoyl-CoA reductases (BCRs) catalyse a key reaction in the anaerobic degradation pathways of monocyclic aromatic substrates, the dearomatization of benzoyl-CoA (BzCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA) at the negative redox potential limit of diffusible enzymatic substrate/product couples (E°' = -622 mV). A 1-MDa class II BCR complex composed of the BamBCDEGHI subunits has so far only been isolated from the Fe(III)-respiring Geobacter metallireducens. It is supposed to drive endergonic benzene ring reduction at an active site W-pterin cofactor by flavin-based electron bifurcation. Here, we identified multiple copies of putative genes encoding the structural components of a class II BCR in sulfate reducing, Fe(III)-respiring and syntrophic bacteria. A soluble 950 kDa Bam[(BC)2 DEFGHI]2 complex was isolated from extracts of Desulfosarcina cetonica cells grown with benzoate/sulfate. Metal and cofactor analyses together with the identification of conserved binding motifs gave rise to 4 W-pterins, two selenocysteines, six flavin adenine dinucleotides, four Zn, and 48 FeS clusters. The complex exhibited 1,5-dienoyl-CoA-, NADPH- and ferredoxin-dependent oxidoreductase activities. Our results indicate that high-molecular class II BCR metalloenzyme machineries are remarkably conserved in strictly anaerobic bacteria with regard to subunit architecture and cofactor content, but their subcellular localization and electron acceptor preference may differ as a result of adaptations to variable energy metabolisms.

Expression of benzoyl-CoA metabolism genes in the lignocellulolytic host Caldicellulosiruptor bescii

Genes responsible for the anaerobic catabolism of benzoate in the thermophilic archaeon Ferroglobus placidus were expressed in the thermophilic lignocellulose-degrading bacterium Caldicellulosiruptor bescii, as a first step to engineering this bacterium to degrade this lignin metabolite. The benzoyl-CoA ligase gene was expressed individually, and in combination with benzoyl-CoA reductase and a putative benzoate transporter. This effort also assessed heterologous expression from a synthetically designed operon whereby each coding sequence was proceeded by a unique C. bescii ribosome binding site sequence. The F. placidicus benzoyl-CoA ligase gene was expressed in C. bescii to produce a full-length protein with catalytic activity. A synthetic 6-gene operon encoding three enzymes involved in benzoate degradation was also successfully expressed in C. bescii as determined by RNA analysis, though the protein products of only four of the genes were detected. The discord between the mRNA and protein measurements, especially considering the two genes lacking apparent protein abundance, suggests variable effectiveness of the ribosome binding site sequences utilized in this synthetic operon. The engineered strains did not degrade benzoate. Although the heterologously expressed gene encoding benzoyl-CoA ligase yielded a protein that was catalytically active in vitro, expression in C. bescii of six benzoate catabolism-related genes combined in a synthetic operon yielded mixed results. More effective expression and in vivo activity might be brought about by validating and using different ribosome binding sites and different promoters. Expressing additional pathway components may alleviate any pathway inhibition and enhance benzoyl-CoA reductase activity.

Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli.

The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.

Stereochemical Insights into the Anaerobic Degradation of 4-Isopropylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1.

The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)-2-hydroxy-4-isopropylcyclohexane-1-carboxylate and (S)-3-isopropylpimelate, of anaerobic degradation of p-cymene in the bacterium Aromatoleum aromaticum pCyN1 are reported. These intermediates (as CoA esters) are involved in the further degradation of 4-isopropylbenzoyl-CoA formed by methyl group hydroxylation and subsequent oxidation of p-cymene. Proteogenomics indicated 4-isopropylbenzoyl-CoA degradation involves (i) a novel member of class I benzoyl-CoA reductase (BCR) as known from Thauera aromatica K172 and (ii) a modified β-oxidation pathway yielding 3-isopropylpimeloyl-CoA analogously to benzoyl-CoA degradation in Rhodopseudomonas palustris. Reference standards of all four diastereoisomers of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate as well as both enantiomers of 3-isopropylpimelate were obtained by stereoselective syntheses via methyl 4-isopropyl-2-oxocyclohexane-1-carboxylate. The stereogenic center carrying the isopropyl group was established using a rhodium-catalyzed asymmetric conjugate addition. X-ray crystallography revealed that the thermodynamically most stable stereoisomer of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate is formed during p-cymene degradation. Our findings imply that the reductive dearomatization of 4-isopropylbenzoyl-CoA by the BCR of A. aromaticum pCyN1 stereospecifically forms (S)-4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA.

QM/MM Study of Tungsten-Dependent Benzoyl-Coenzyme A Reductase: Rationalization of Regioselectivity and Predication of W vs Mo Selectivity

The class II benzoyl-coenzyme A reductase (BCR) is a tungsten-dependent enzyme that catalyzes the Birch reduction of benzoyl-CoA to a cyclic diene. The reaction mechanism and regioselectivity of benzoyl-CoA were explored through QM/MM calculations using two different QM regions (124 atoms and 223 atoms) on the solvated enzyme. The reduction reaction involves two major chemical steps that both proceed in the triplet state or in the broken-symmetry singlet state. First, the tungsten-bound water molecule delivers a proton to the C4 of the benzoyl-CoA substrate, coupled with an electron transfer from the WIV center to the substrate. This leads to the formation of a WV-radical intermediate, with a barrier of 23.2 kcal/mol in the broken-symmetry singlet state at the B3LYP-D3/def2-TZVPP:Charmm level. Subsequently, the protonated His260 residue delivers a second proton to C3 of the benzoyl-CoA substrate, concomitantly with the shift of the second electron from the pyranopterin cofactor rather than the WV ion to the substrate, which has a barrier of 19.1 kcal/mol at the B3LYP-D3/def2-TZVPP:Charmm level and produces the cyclohexa-1,5-diene-1-carboxyl-CoA product. The reduction of the aromatic ring at other positions has also been considered; however, the barriers are much higher, which shows that the cyclohexa-1,5-diene-1-carboxyl-CoA product is exclusively formed during the benzoyl-CoA reduction. Moreover, molybdenum, tungsten's lighter congener, has also been considered to replace the tungsten ion in the benzoyl-CoA reductase. The molybdenum substituted enzyme (Mo-BCR) was found to have a quite higher barrier for the reduction reaction, but a feasible barrier for the reverse oxidation reaction.

A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of methyl- and halobenzoates in denitrifying bacteria

Class I benzoyl-CoA (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of the central BzCoA intermediate and analogues of it to conjugated cyclic 1,5-dienoyl-CoAs probably by a radical-based, Birch-like reduction mechanism. Discovered in 1995, the enzyme from the denitrifying bacterium Thauera aromatica (BCRTar) has so far remained the only isolated and biochemically accessible BCR, mainly because BCRs are extremely labile, and their heterologous production has largely failed so far. Here, we describe a platform for the heterologous expression of the four structural genes encoding a designated 3-methylbenzoyl-CoA reductase from the related denitrifying species Thauera chlorobenzoica (MBRTcl) in Escherichia coli This reductase represents the prototype of a distinct subclass of ATP-dependent BCRs that were proposed to be involved in the degradation of methyl-substituted BzCoA analogues. The recombinant MBRTcl had an αβγδ-subunit architecture, contained three low-potential [4Fe-4S] clusters, and was highly oxygen-labile. It catalyzed the ATP-dependent reductive dearomatization of BzCoA with 2.3-2.8 ATPs hydrolyzed per two electrons transferred and preferentially dearomatized methyl- and chloro-substituted analogues in meta- and para-positions. NMR analyses revealed that 3-methylbenzoyl-CoA is regioselectively reduced to 3-methyl-1,5-dienoyl-CoA. The unprecedented reductive dechlorination of 4-chloro-BzCoA to BzCoA probably via HCl elimination from a reduced intermediate allowed for the previously unreported growth of T. chlorobenzoica on 4-chlorobenzoate. The heterologous expression platform established in this work enables the production, isolation, and characterization of bacterial and archaeal BCR and BCR-like radical enzymes, for many of which the function has remained unknown.

One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window

Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to -0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; however, the mechanism and components involved in the energetic coupling of endergonic benzene ring reduction have remained hypothetical. We present a 1-MDa, membrane-associated, Bam[(BC)2DEFGHI]2 complex from the anaerobic bacterium Geobacter metallireducens harboring 4 tungsten, 4 zinc, 2 selenocysteines, 6 FAD, and >50 FeS cofactors. The results suggest that class II BCRs catalyze electron transfer to the aromatic ring, yielding a cyclic 1,5-dienoyl-CoA via two flavin-based electron bifurcation events. This work expands our knowledge of energetic couplings in biology by high-molecular-mass electron bifurcating machineries.

Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes.

ABSTRACTThe deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive dehalogenation, as well as the biology of abundant deep-sea Chloroflexi.

Breaking Benzene Aromaticity-Computational Insights into the Mechanism of the Tungsten-Containing Benzoyl-CoA Reductase.

Aromatic compounds are environmental pollutants with toxic and carcinogenic properties. Despite the stability of aromatic rings, bacteria are able to degrade the aromatic compounds into simple metabolites and use them as growth substrates under oxic or even under anoxic conditions. In anaerobic microorganisms, most monocyclic aromatic growth substrates are converted to the central intermediate benzoyl-coenzyme A, which is enzymatically reduced to cyclohexa-1,5-dienoyl-CoA. The strictly anaerobic bacterium Geobacter metallireducens uses the class II benzoyl-CoA reductase complex for this reaction. The catalytic BamB subunit of this complex harbors an active site tungsten-bis-pyranopterin cofactor with the metal being coordinated by five protein/cofactor-derived sulfur atoms and a sixth, so far unknown, ligand. Although BamB has been biochemically and structurally characterized, its mechanism still remains elusive. Here we use continuum electrostatic and QM/MM calculations to model benzoyl-CoA reduction by BamB. We aim to elucidate the identity of the sixth ligand of the active-site tungsten ion together with the interplay of the electron and proton transfer events during the aromatic ring reduction. On the basis of our calculations, we propose that benzoyl-CoA reduction is initiated by a hydrogen atom transfer from a W(IV) species with an aqua ligand, yielding W(V)-[OH-] and a substrate radical intermediate. In the next step, a proton-assisted second electron transfer takes place with a conserved active-site histidine serving as the second proton donor. Interestingly, our calculations suggest that the electron for the second reduction step is taken from the pyranopterin cofactors rather than from the tungsten ion. The resulting cationic radical, which is distributed over both pyranopterins, is stabilized by conserved anionic amino acid residues. The stepwise mechanism of the reduction shows similarities to the Birch reduction known from organic chemistry. However, the strict coupling of protons and electrons allows the reaction to proceed under milder conditions.

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model.

Previously, Planted Fixed-Bed Reactors (PFRs) have been used to investigate microbial toluene removal in the rhizosphere of constructed wetlands. Aerobic toluene degradation was predominant in these model systems although bulk redox conditions were hypoxic to anoxic. However, culture-independent approaches indicated also that microbes capable of anaerobic toluene degradation were abundant. Therefore, we aimed at isolating anaerobic-toluene degraders from one of these PFRs. From the obtained colonies which consisted of spirilli-shaped bacteria, a strain designated 15–1 was selected for further investigations. Analysis of its 16S rRNA gene revealed greatest similarity (99%) with toluene-degrading Magnetospirillum sp. TS-6. Isolate 15–1 grew with up to 0.5 mM of toluene under nitrate-reducing conditions. Cells reacted to higher concentrations of toluene by an increase in the degree of saturation of their membrane fatty acids. Strain 15–1 contained key genes for the anaerobic degradation of toluene via benzylsuccinate and subsequently the benzoyl-CoA pathway, namely bssA, encoding for the alpha subunit of benzylsuccinate synthase, bcrC for subunit C of benzoyl-CoA reductase and bamA for 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase. Finally, most members of a clone library of bssA generated from the PFR had highest similarity to bssA from strain 15–1. Our study provides insights about the physiological capacities of a strain of Magnetospirillum isolated from a planted system where active rhizoremediation of toluene is taking place.