benzoyl-CoA Reductase Research Articles

Reconstructing metabolic pathways of a member of the genus Pelotomaculum suggesting its potential to oxidize benzene to carbon dioxide with direct reduction of sulfate.

The enrichment culture BPL is able to degrade benzene with sulfate as electron acceptor and is dominated by an organism of the genus Pelotomaculum. Members of Pelotomaculum are usually known to be fermenters, undergoing syntrophy with anaerobic respiring microorganisms or methanogens. By using a metagenomic approach, we reconstructed a high-quality genome (∼2.97 Mbp, 99% completeness) for Pelotomaculum candidate BPL. The proteogenomic data suggested that (1) anaerobic benzene degradation was activated by a yet unknown mechanism for conversion of benzene to benzoyl-CoA; (2) the central benzoyl-CoA degradation pathway involved reductive dearomatization by a class II benzoyl-CoA reductase followed by hydrolytic ring cleavage and modified β-oxidation; (3) the oxidative acetyl-CoA pathway was utilized for complete oxidation to CO2. Interestingly, the genome of Pelotomaculum candidate BPL has all the genes for a complete sulfate reduction pathway including a similar electron transfer mechanism for dissimilatory sulfate reduction as in other Gram-positive sulfate-reducing bacteria. The proteome analysis revealed that the essential enzymes for sulfate reduction were all formed during growth with benzene. Thus, our data indicated that, besides its potential to anaerobically degrade benzene, Pelotomaculum candidate BPL is the first member of the genus that can perform sulfate reduction.

Spatial Abundance and Distribution of Potential Microbes and Functional Genes Associated with Anaerobic Mineralization of Pentachlorophenol in a Cylindrical Reactor.

Functional interplays of microbial activity, genetic diversity and contaminant transformation are poorly understood in reactors for mineralizing halogenated aromatics anaerobically. Here, we investigated abundance and distribution of potential microbes and functional genes associated with pentachlorophenol (PCP) anaerobic mineralization in a continuous-flow cylindrical reactor (15 cm in length). PCP dechlorination and the metabolite (phenol) were observed at segments 0–8 cm from inlet, where key microbes, including potential reductive dechlorinators (Dehalobacter, Sulfurospirillum, Desulfitobacterium and Desulfovibrio spp.) and phenol degraders (Cryptanaerobacter and Syntrophus spp.), as well as putative functional genes, including putative chlorophenol reductive dehalogenase (cprA) and benzoyl-CoA reductase (bamB), were highly enriched simultaneously. Five types of putative cprAs, three types of putative bamBs and seven types of putative nitrogenase reductase (nifHs) were determined, with their copy numbers decreased gradually from inlet to outlet. Distribution of chemicals, bacteria and putative genes confirmed PCP dechlorination and phenol degradation accomplished in segments 0–5 cm and 0–8 cm, respectively, contributing to a high PCP mineralization rate of 3.86 μM d−1. Through long-term incubation, dechlorination, phenol degradation and nitrogen fixation bacteria coexisted and functioned simultaneously near inlet (0–8 cm), verified the feasibility of anaerobic mineralization of halogenated aromatics in the compact reactor containing multiple functional microbes.

Anaerobic Degradation of p-Alkylated Benzoates and Toluenes

The anaerobic degradation of 4-alkylbenzoates and 4-alkyltoluenes is to date a rarely reported microbial capacity. The newly isolated Alphaproteobacterium Magnetospirillum sp. strain pMbN1 represents the first pure culture demonstrated to degrade 4-methylbenzoate completely to CO₂ in a process coupled to denitrification. Differential proteogenomic studies in conjunction with targeted metabolite analyses and enzyme activity measurements elucidated a specific 4-methylbenzoyl-coenzyme A (CoA) pathway in this bacterium alongside the classical central benzoyl-CoA pathway. Whilst these two pathways are analogous, in the former the p-methyl group is retained and its 4-methylbenzoyl-CoA reductase (MbrCBAD) is phylogenetically distinct from the archetypical class I benzoyl-CoA reductase (BcrCBAD). Subsequent global regulatory studies on strain pMbN1 grown with binary or ternary substrate mixtures revealed benzoate to repress the anaerobic utilization of 4-methylbenzoate and succinate. The shared nutritional property of betaproteobacterial ‘Aromatoleum aromaticum' pCyN1 and Thauera sp. strain pCyN2 is the anaerobic degradation of the plant-derived hydrocarbon p-cymene (4-isopropyltoluene) coupled to denitrification. Notably, the two strains employ two different peripheral pathways for the conversion of p-cymene to 4-isopropylbenzoyl-CoA as the possible first common intermediate. In ‘A. aromaticum' pCyN1 a putative p-cymene dehydrogenase (CmdABC) is proposed to hydroxylate the benzylic methyl group, which is subsequently further oxidized to the CoA-thioester. In contrast, Thauera sp. strain pCyN2 employs a reaction sequence analogous to the known anaerobic toluene pathway, involving a distinct branching (4-isopropylbenzyl)succinate synthase (IbsABCDEF).

Enzymes of the benzoyl-coenzyme A degradation pathway in the hyperthermophilic archaeon Ferroglobus placidus.

The Fe(III)-respiring Ferroglobus placidus is the only known archaeon and hyperthermophile for which a complete degradation of aromatic substrates to CO2 has been reported. Recent genome and transcriptome analyses proposed a benzoyl-coenzyme A (CoA) degradation pathway similar to that found in the phototrophic Rhodopseudomonas palustris, which involves a cyclohex-1-ene-1-carboxyl-CoA (1-enoyl-CoA) forming, ATP-dependent key enzyme benzoyl-CoA reductase (BCR). In this work, we demonstrate, by first in vitro studies, that benzoyl-CoA is ATP-dependently reduced by two electrons to cyclohexa-1,5-dienoyl-CoA (1,5-dienoyl-CoA), which is further degraded by hydration to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA (6-OH-1-enoyl-CoA); upon addition of NAD(+) , the latter was subsequently converted to β-oxidation intermediates. The four candidate genes of BCR were heterologously expressed, and the enriched, oxygen-sensitive enzyme catalysed the two-electron reduction of benzoyl-CoA to 1,5-dienoyl-CoA. A gene previously assigned to a 2,3-didehydropimeloyl-CoA hydratase was heterologously expressed and shown to act as a typical 1,5-dienoyl-CoA hydratase that does not accept 1-enoyl-CoA. A gene previously assigned to a 1-enoyl-CoA hydratase was heterologously expressed and identified to code for a bifunctional crotonase/3-OH-butyryl-CoA dehydrogenase. In summary, the results consistently provide biochemical evidence that F. placidus and probably other archaea predominantly degrade aromatics via the Thauera/Azoarcus type and not or only to a minor extent via the predicted R. palustris-type benzoyl-CoA degradation pathway.

The benzoyl-coenzyme a reductase and 2-hydroxyacyl-coenzyme a dehydratase radical enzyme family.

Benzoyl-CoA reductases (BCRs) and 2-hydroxyacyl-CoA dehydratases (HAD) contain Fe–S clusters and catalyse two (at first glance, unrelated) mechanistically difficult reactions. The common feature of this family is a stoichiometric (BCR) or catalytic (HAD) ATP hydrolysis; this drives electron transfer from a ferredoxin to a CoA thioester, thus generating an extremely low-potential ketyl radical.

Metaproteogenomic analysis of a sulfate-reducing enrichment culture reveals genomic organization of key enzymes in the m-xylene degradation pathway and metabolic activity of proteobacteria

This study aimed to ascertain the functional and phylogenetic relationships within an m-xylene degrading sulfate-reducing enrichment culture, which had been maintained for several years in the laboratory with m-xylene as the sole source of carbon and energy. Previous studies indicated that a phylotype affiliated to the Desulfobacteraceae was the main m-xylene assimilating organism. In the present study, genes and gene products were identified by a metaproteogenomic approach using LC-MS/MS analysis of the microbial community, and 2426 peptides were identified from 576 proteins. In the metagenome of the community, gene clusters encoding enzymes involved in fumarate addition to a methyl moiety of m-xylene (nms, bss), as well as gene clusters coding for enzymes involved in modified beta-oxidation to (3-methyl)benzoyl-CoA (bns), were identified in two separate contigs. Additionally, gene clusters containing homologues to bam genes encoding benzoyl-CoA reductase (Bcr) class II, catalyzing the dearomatization of (3-methyl)benzoyl-CoA, were identified. Time-resolved protein stable isotope probing (protein-SIP) experiments using 13C-labeled m-xylene showed that the respective gene products were highly 13C-labeled. The present data suggested the identification of gene products that were similar to those involved in methylnaphthalene degradation even though the consortium was not capable of growing in the presence of naphthalene, methylnaphthalene or toluene as substrates. Thus, a novel branch of enzymes was found that was probably specific for anaerobic m-xylene degradation.

Chapter Five - Benzoyl-CoA, a Universal Biomarker for Anaerobic Degradation of Aromatic Compounds

Aromatic compounds are a major component of the global carbon pool and include a diverse range of compounds such as humic acid, lignin, amino acids, and industrial contaminants. Due to the prevalence of aromatic compounds in the environment, aerobic and anaerobic microorganisms have evolved mechanisms by which to metabolize that available carbon. Less well understood are the anaerobic pathways. We now know that anaerobic metabolism of a variety of monoaromatic compounds can be initiated in a number of different ways, and a key metabolite for these pathways is benzoyl-CoA. Chemicals can have different upstream anaerobic degradation pathways yet can still be assessed by targeting the downstream benzoyl-CoA pathway. In this pathway, we propose that the ring opening hydrolase, encoded by the bamA gene, is especially useful because, in contrast to the benzoyl-CoA reductase, it is detected under a number of respiratory settings, including denitrifying, iron-reducing, sulfate-reducing, and fermentative conditions, and has a wide distribution in the environment. This review examines the bamA gene in enrichment cultures and environmental DNA extracts to consider whether it can be used as a biomarker for anaerobic aromatic degradation. Given the number of potential upstream inputs from natural and man-made monoaromatic compounds, the benzoyl-CoA pathway and the bamA gene in particular may play an important role in the global carbon cycle that has thus far been overlooked.

Metabolic versatility of toluene‐degrading, iron‐reducing bacteria in tidal flat sediment, characterized by stable isotope probing‐based metagenomic analysis

DNA stable isotope probing and metagenomic sequencing were used to assess the metabolic potential of iron-reducing bacteria involved in anaerobic aromatic hydrocarbon degradation in oil spill-affected tidal flats. In a microcosm experiment, (13) C-toluene was degraded with the simultaneous reduction of Fe(III)-NTA, which was also verified by quasi-stoichiometric (13) C-CO2 release. The metabolic potential of the dominant member affiliated with the genus Desulfuromonas in the heavy DNA fraction was inferred using assembled scaffolds (designated TF genome, 4.40 Mbp with 58.8 GC mol%), which were obtained by Illumina sequencing. The gene clusters with peripheral pathways for toluene and benzoate conversion possessed the features of strict and facultative anaerobes. In addition to the class II-type benzoyl-CoA reductase (Bam) of strict anaerobes, the class I-type (Bcr) of facultative anaerobes was encoded. Genes related to the utilization of various anaerobic electron acceptors, including iron, nitrate (to ammonia), sulfur and fumarate, were identified. Furthermore, genes encoding terminal oxidases (caa3 , cbb3 and bd) and a diverse array of genes for oxidative stress responses were detected in the TF genome. This metabolic versatility may be an adaptation to the fluctuating availability of electron acceptors and donors in tidal flats.

Cyclohexanecarboxyl-coenzyme A (CoA) and cyclohex-1-ene-1-carboxyl-CoA dehydrogenases, two enzymes involved in the fermentation of benzoate and crotonate in Syntrophus aciditrophicus.

The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.

Identification and characterization of 2‐naphthoyl‐coenzyme A reductase, the prototype of a novel class of dearomatizing reductases

The 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 benzoyl-coenzyme 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.

ATP‐dependent/‐independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2-methylnaphthalene in the sulfate-reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite-dependent four-electron reduction of the key intermediate 2-naphthoyl-coenzyme A (NCoA) to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two-electron reaction to most likely a conjugated hexahydro-2-naphthoyl-CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen-sensitive with a half-life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP-dependent class I benzoyl-CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl-carboxyl-CoA reductases.

A Depth Resolved Insight into Benzoyl CoA Reductase and Benzoate Dioxygenase Gene Copy Numbers within a Marine Sediment Associated with Methane Seepage

A marine sediment core obtained from a methane seepage site off the northern coast of Irelandwas analysed, at 3 depths, for catabolic genes associated with the aerobic and anaerobic degradation of aromatic compounds. Catabolic gene copy numbers varied through the core peaking at 2.1 meters below sediment surface (mbsf)-just above the sulphate boundary. Beyond the sulphate boundary gene copy numbers fell considerably, suggesting the boundary may be a critical factor in the degradation of aromatic compounds within marine sediments. At the 2.1 mbsf depth our data also suggest that known benzoyl CoA reductase utilizing bacteria are readily detectable.

Complete genome, catabolic sub‐proteomes and key‐metabolites of Desulfobacula toluolica Tol2, a marine, aromatic compound‐degrading, sulfate‐reducing bacterium

Among the dominant deltaproteobacterial sulfate-reducing bacteria (SRB), members of the genus Desulfobacula are not only present in (hydrocarbon-rich) marine sediments, but occur also frequently in the anoxic water bodies encountered in marine upwelling areas. Here, we present the 5.2 Mbp genome of Desulfobacula toluolica Tol2, which is the first of an aromatic compound-degrading, marine SRB. The genome has apparently been shaped by viral attacks (e.g. CRISPRs) and its high plasticity is reflected by 163 detected genes related to transposases and integrases, a total of 494 paralogous genes and 24 group II introns. Prediction of the catabolic network of strain Tol2 was refined by differential proteome and metabolite analysis of substrate-adapted cells. Toluene and p-cresol are degraded by separate suites of specific enzymes for initial arylsuccinate formation via addition to fumarate (p-cresol-specific enzyme HbsA represents a new phylogenetic branch) as well as for subsequent modified β-oxidation of arylsuccinates to the central intermediate benzoyl-CoA. Proteogenomic evidence suggests specific electron transfer (EtfAB) and membrane proteins to channel electrons from dehydrogenation of both arylsuccinates directly to the membrane redox pool. In contrast to the known anaerobic degradation pathways in other bacteria, strain Tol2 deaminates phenylalanine non-oxidatively to cinnamate by phenylalanine ammonia-lyase and subsequently forms phenylacetate (both metabolites identified in (13) C-labelling experiments). Benzoate degradation involves CoA activation, reductive dearomatization by a class II benzoyl-CoA reductase and hydrolytic ring cleavage as found in the obligate anaerobe Geobacter metallireducens GS-15. The catabolic sub-proteomes displayed high substrate specificity, reflecting the genomically predicted complex and fine-tuned regulatory network of strain Tol2. Despite the genetic equipment for a TCA cycle, proteomic evidence supports complete oxidation of acetyl-CoA to CO2 via the Wood-Ljungdahl pathway. Strain Tol2 possesses transmembrane redox complexes similar to that of other Desulfobacteraceae members. The multiple heterodisulfide reductase-like proteins (more than described for Desulfobacterium autotrophicum HRM2) may constitute a multifaceted cytoplasmic electron transfer network.

Characterization of the mbd cluster encoding the anaerobic 3‐methylbenzoyl‐CoA central pathway

The mbd cluster encoding genes of the 3-methylbenzoyl-CoA pathway involved in the anaerobic catabolism of 3-methylbenzoate and m-xylene was characterized for the first time in the denitrifying β-Proteobacterium Azoarcus sp. CIB. The mbdA gene product was identified as a 3-methylbenzoate-CoA ligase required for 3-methylbenzoate activation; its substrate spectrum was unique in activating all three methylbenzoate isomers. An inducible 3-methylbenzoyl-CoA reductase (mbdONQP gene products), displaying significant amino acid sequence similarities to known class I benzoyl-CoA reductases catalysed the ATP-dependent reduction of 3-methylbenzoyl-CoA to a methyldienoyl-CoA. The mbdW gene encodes a methyldienoyl-CoA hydratase that hydrated the methyldienoyl-CoA to a methyl-6-hydroxymonoenoyl-CoA compound. The mbd cluster also contains the genes predicted to be involved in the subsequent steps of the 3-methylbenzoyl-CoA pathway as well as the electron donor system for the reductase activity. Whereas the catabolic mbd genes are organized in two divergent inducible operons, the putative mbdR regulatory gene was transcribed separately and showed constitutive expression. The efficient expression of the mbd genes required the oxygen-dependent AcpR activator, and it was subject of carbon catabolite repression by some organic acids and amino acids. Sequence analyses suggest that the mbd gene cluster was recruited by Azoarcus sp. CIB through horizontal gene transfer.

Anaerobic degradation of 4‐methylbenzoate via a specific 4‐methylbenzoyl‐CoA pathway

The pathway for anaerobic degradation of 4-methylbenzoate was studied in the denitrifying alphaproteobacterium Magnetospirillum sp. strain pMbN1. Adaptation studies with whole cells indicated substrate-dependent induction of the capacity to degrade 4-methylbenzoate. Differential protein profiling (2D-DIGE) of 4-methylbenzoate- in comparison with benzoate- or succinate-adapted cells revealed the specific abundance increase of substrate-specific protein sets. Their coding genes form distinct clusters on the genome, two of which were assigned to 4-methylbenzoate and one to benzoate degradation. The predicted functions of the gene products agree with a specific 4-methylbenzoyl-CoA degradation pathway in addition to and analogous to the known anaerobic benzoyl-CoA degradation pathway. In vitro benzoyl-CoA and 4-methylbenzoyl-CoA reductase activities revealed the electron donor and ATP-dependent formation of the corresponding conjugated cyclic dienoyl-CoA/4-methyl-dienoyl-CoA products. The 4-methylbenzoyl-CoA reductase activity was induced in the presence of 4-methylbenzoate. In accordance, metabolite analysis of cultures grown with 4-methylbenzoate tentatively identified 4-methylcyclohex-1,5-diene-1-carboxylate. The 4-methylbenzoate induced genes were assigned to code for the putative 4-methylbenzoyl-CoA reductase; their products display pronounced sequence disparity from the conventional class I benzoyl-CoA reductase, which does not accept substituents at the para-position. Identification of 3-methylglutarate together with the formation of specific proteins for ring cleavage and β-oxidation in 4-methylbenzoate-adapted cells suggest conservation of the methyl group along the specific 4-methylbenzoyl-CoA degradation pathway.

Enzymes involved in the anaerobic degradation of meta‐substituted halobenzoates

Organohalides are environmentally relevant compounds that can be degraded by aerobic and anaerobic microorganisms. The denitrifying Thauera chlorobenzoica is capable of degrading halobenzoates as sole carbon and energy source under anaerobic conditions. LC-MS/MS-based coenzyme A (CoA) thioester analysis revealed that 3-chloro- or 3-bromobenzoate were preferentially metabolized via non-halogenated CoA-ester intermediates of the benzoyl-CoA degradation pathway. In contrast, 3-fluorobenzoate, which does not support growth, was converted to dearomatized fluorinated CoA ester dead-end products. Extracts from cells grown on 3-chloro-/3-bromobenzoate catalysed the Ti(III)-citrate- and ATP-dependent reductive dehalogenation of 3-chloro/3-bromobenzoyl-CoA to benzoyl-CoA, whereas 3-fluorobenzoyl-CoA was converted to a fluorinated cyclic dienoyl-CoA compound. The reductive dehalogenation reactions were identified as previously unknown activities of ATP-dependent class I benzoyl-CoA reductases (BCR) present in all facultatively anaerobic, aromatic compound degrading bacteria. A two-step dearomatization/H-halide elimination mechanism is proposed. A halobenzoate-specific carboxylic acid CoA ligase was characterized in T. chlorobenzoica; however, no such enzyme is present in Thauera aromatica, which cannot grow on halobenzoates. In conclusion, it appears that the presence of a halobenzoate-specific carboxylic acid CoA ligase rather than a specific reductive dehalogenase governs whether an aromatic compound degrading anaerobe is capable of metabolizing halobenzoates.

Genome-scale analysis of anaerobic benzoate and phenol metabolism in the hyperthermophilic archaeon Ferroglobus placidus.

Insight into the mechanisms for the anaerobic metabolism of aromatic compounds by the hyperthermophilic archaeon Ferroglobus placidus is expected to improve understanding of the degradation of aromatics in hot (>80° C) environments and to identify enzymes that might have biotechnological applications. Analysis of the F. placidus genome revealed genes predicted to encode enzymes homologous to those previously identified as having a role in benzoate and phenol metabolism in mesophilic bacteria. Surprisingly, F. placidus lacks genes for an ATP-independent class II benzoyl-CoA (coenzyme A) reductase (BCR) found in all strictly anaerobic bacteria, but has instead genes coding for a bzd-type ATP-consuming class I BCR, similar to those found in facultative bacteria. The lower portion of the benzoate degradation pathway appears to be more similar to that found in the phototroph Rhodopseudomonas palustris, than the pathway reported for all heterotrophic anaerobic benzoate degraders. Many of the genes predicted to be involved in benzoate metabolism were found in one of two gene clusters. Genes for phenol carboxylation proceeding through a phenylphosphate intermediate were identified in a single gene cluster. Analysis of transcript abundance with a whole-genome microarray and quantitative reverse transcriptase polymerase chain reaction demonstrated that most of the genes predicted to be involved in benzoate or phenol metabolism had higher transcript abundance during growth on those substrates vs growth on acetate. These results suggest that the general strategies for benzoate and phenol metabolism are highly conserved between microorganisms living in moderate and hot environments, and that anaerobic metabolism of aromatic compounds might be analyzed in a wide range of environments with similar molecular targets.

Structural Basis for Reductive Radical Formation and Electron Recycling in (R)-2-Hydroxyisocaproyl-CoA Dehydratase

The radical enzyme (R)-2-hydroxyisocaproyl-CoA dehydratase catalyzes the dehydration of (R)-2-hydroxyisocaproyl-CoA in the fermentation of l-leucine by the human pathogenic bacterium Clostridium difficile. In contrast to other radical enzymes, such as bacterial class II ribonucleotide reductase or biotin synthase, the Fe/S cluster containing (R)-2-hydroxyisocaproyl-CoA dehydratase requires no special cofactors such as coenzyme B(12) or S-adenosylmethionine for radical generation. Instead it uses a single high-energy electron that is recycled after each turnover. The catalyzed reaction, an atypical α/β-dehydration, depends on the reductive formation of ketyl radicals on the substrate generated by injection of a single electron from the ATP-dependent activator protein. So far, it is unknown how the active electron is recycled and how unwanted side reactions are prevented, allowing for up to 10,000 turnovers. The crystal structure reveals that the heterodimeric protein contains two [4Fe-4S] clusters at a distance of 12 Å, each coordinated by three cysteines and one terminal ligand. The cluster in the α-subunit is part of the active site. In the absence of substrate, a water/hydroxide ion acts as the fourth ligand. The substrate replaces this ligand and coordinates the cluster via the carbonyl-oxygen of the thioester group. The cluster in the β-subunit has a terminal sulfhydryl/sulfido ligand and can act as a reservoir to protect the electron from unwanted side reactions via a recycling mechanism. The crystal structure of (R)-2-hydroxyisocaproyl-CoA dehydratase serves as a model for the reductively radical-generating metalloenzymes of the (R)-2-hydroxyacyl-CoA dehydratase and benzoyl-CoA reductase families.

A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa

BackgroundFerredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.ResultsA bioinformatic screening of the sequence features defining very low potential 2[4Fe-4S] ferredoxins has revealed the almost exclusive presence of the corresponding fdx gene in the Proteobacteria phylum, without occurrence in Archaea and Eukaryota. The transcript was found to be monocistronic in Pseudomonas aeruginosa, and not part of an operon in most bacteria. Only fdx genes of bacteria which anaerobically degrade aromatic compounds belong to operons. As this pathway is not present in all bacteria having very low potential 2[4Fe-4S] ferredoxins, these proteins cannot exclusively be reductants of benzoyl CoA reductases. Expression of the ferredoxin gene did not change in response to varying growth conditions, including upon macrophage infection or aerobic growth with 4-hydroxy benzoate as carbon source. However, it increased along the growth curve in Pseudomonas aeruginosa and in Escherichia coli. The sequence immediately 5' upstream of the coding sequence contributed to the promotor activity. Deleting the fdx gene in Pseudomonas aeruginosa abolished growth, unless a plasmid copy of the gene was provided to the deleted strain.ConclusionsThe gene of the very low potential 2[4Fe-4S] ferredoxin displays characteristics of a housekeeping gene, and it belongs to the minority of genes that are essential in Pseudomonas aeruginosa. These data identify a new potential antimicrobial target in this and other pathogenic Proteobacteria.

Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO(2). The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of alpha-, beta-, and gamma-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe(4)S(4)] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers "Aromatoleum aromaticum" EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO(2). Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.