Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in 'Birch-like' reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., -1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.     

Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in ‘Birch-like’ reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., −1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.     

Dearomatizing benzene ring reductases

The high resonance energy of the benzene ring is responsible for the relative resistance of aromatic compounds to biodegradation. Nevertheless, bacteria from nearly all physiological groups have been isolated which utilize aromatic growth substrates as the sole source of cell carbon and energy. The enzymatic dearomatization of the benzene nucleus by microorganisms is accomplished in two different manners. In aerobic bacteria the aromatic ring is dearomatized by oxidation, catalyzed by oxygenases. In contrast, anaerobic bacteria attack the aromatic ring by reductive steps. Key intermediates in the anaerobic aromatic metabolism are benzoyl-CoA and compounds with at least two meta-positioned hydroxyl groups (resorcinol, phloroglucinol and hydroxyhydroquinone). In facultative anaerobes, the reductive dearomatization of the key intermediate benzoyl-CoA requires a stoichiometric coupling to ATP hydrolysis, whereas reduction of the other intermediates is readily achieved with suitable electron donors. Obligately anaerobic bacteria appear to use a totally different enzymology for the reductive dearomatization of benzoyl-CoA including selenocysteine- and molybdenum- containing enzymes.     

Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.     

Reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA in a denitrifying, phenol-degrading Pseudomonas species

The initial reactions in anaerobic degradation of phenol to CO2 have been studied in vitro with a denitrifying Pseudomonas strain grown with phenol and nitrate in the absence of molecular oxygen. Phenol has been proposed to be carboxylated to 4-hydroxybenzoate [(1987) Arch. Microbiol. 148, 213-217]. 4-Hydroxybenzoate was activated to 4-hydroxybenzoyl-CoA by a coenzyme A ligase. Cell extracts also catalyzed the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA with reduced benzyl viologen as electron donor. This enzyme, benzoyl-CoA:(acceptor) 4-oxidoreductase (hydroxylating) (EC 1.3.99.-), has not been reported before. The data suggest that phenol and 4-hydroxybenzoate are anaerobically metabolized by this strain via benzoyl-CoA.     

Anaerobic degradation of catechol by Desulfobacterium sp. strain Cat2 proceeds via carboxylation to protocatechuate.

Under anoxic conditions, most methoxylated mononuclear aromatic compounds are degraded by bacteria, with catechol being formed as an important intermediate. On the basis of our experiments with the sulfate-reducing bacterium Desulfobacterium sp. strain Cat2, we describe for the first time the enzymatic activities involved in the complete anaerobic oxidation of catechol and protocatechuate. Results obtained from experiments with dense cell suspensions of strain Cat2 demonstrated that all enzymes necessary for protocatechuate and benzoate degradation were induced during growth with catechol. In addition, anaerobic oxidation of catechol was found to be a CO2-dependent process. Phenol was not degraded in suspensions of cells grown with catechol. In cell extracts of Desulfobacterium sp. strain Cat2, protocatechuyl-coenzyme A (CoA) was formed from catechol, bicarbonate, and uncombined CoA. This oxygen-sensitive reaction requires high concentrations of both bicarbonate and protein, and only very low levels of enzyme were detected. In a second oxygen-sensitive step, protocatechuyl-CoA was reduced to 3-hydroxybenzoyl-CoA by reductive elimination of the p-hydroxyl group. Further dehydroxylation to benzoyl-CoA was not detectable. Key reactions described for anaerobic degradation of benzoate were catalyzed by cell extracts of strain Cat2, too.     

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica

Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent β-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of β-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO₂. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases. Received: 22 December 1997 / Accepted: 12 May 1998     

Occurrence, genes and expression of the W/Se-containing class II benzoyl-coenzyme A reductases in anaerobic bacteria

Benzoyl-coenzyme A (CoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds and catalyse the reductive dearomatization of benzoyl-CoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA. Class I BCRs are ATP-dependent FeS enzymes, whereas class II BCRs are supposed to be ATP-independent and contain W, FeS clusters, and most probably selenocysteine. The active site components of a putative eight subunit class II BCR, BamBCDEFGHI, were recently characterized in Geobacter metallireducens. In this organism bamB was identified as structural gene for the W-containing active site subunit; bamF was predicted to code for a selenocysteine containing electron transfer subunit. In this work the occurrence and expression of BCRs in a number of anaerobic, aromatic compound degrading model microorganisms was investigated with a focus on the BamB and BamF components. Benzoate-induced class II BCR in vitro activities were determined in the soluble protein fraction in all obligately anaerobic bacteria tested. Where applicable, the results were in agreement with Western blot analysis using BamB targeting antibodies. By establishing a specific bamB targeting PCR assay, bamB homologues were identified in all tested obligately anaerobic bacteria with the capacity to degrade aromatic compounds; a number of bamB sequences from Gram-negative/positive sulfate-reducing bacteria were newly sequenced. In several organisms at least two bamB paralogues per genome were identified; however, in nearly all cases only one of them was transcribed during growth on an aromatic substrate. These benzoate-induced bamB genes are proposed to code for the active site subunit of class II BCRs; the major part of them group into a phylogenetic subcluster within the bamB homologues. Results from in silico analysis suggested that all class II BCRs contain selenocysteine in the BamF, and in many cases also in the BamE subunit. The results obtained indicate that the distribution of the two classes of BCRs in anaerobic bacteria appears to be strictly ruled by the available free energy from the oxidation of the aromatic carbon source rather than by phylogenetic relationships.     

Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium.

The enzymes catalyzing the initial reactions in the anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) were studied with a denitrifying Pseudomonas sp. anaerobically grown with 2-aminobenzoate and nitrate as the sole carbon and energy sources. Cells grown on 2-aminobenzoate are simultaneously adapted to growth with benzoate, whereas cells grown on benzoate degrade 2-aminobenzoate several times less efficiently than benzoate. Evidence for a new reductive pathway of aromatic metabolism and for four enzymes catalyzing the initial steps is presented. The organism contains 2-aminobenzoate-coenzyme A ligase (2-aminobenzoate-CoA ligase), which forms 2-aminobenzoyl-CoA. 2-Aminobenzoyl-CoA is then reductively deaminated to benzoyl-CoA by an oxygen-sensitive enzyme, 2-aminobenzoyl-CoA reductase (deaminating), which requires a low potential reductant [Ti(III)]. The specific activity is 15 nmol of 2-aminobenzoyl-CoA reduced min-1 mg-1 of protein at an optimal pH of 7. The two enzymes are induced by the substrate under anaerobic conditions only. Benzoyl-CoA is further converted in vitro by reduction with Ti(III) to six products; the same products are formed when benzoyl-CoA or 2-aminobenzoyl-CoA is incubated under reducing conditions. Two of them were identified preliminarily. One product is cyclohex-1-enecarboxyl-CoA, the other is trans-2-hydroxycyclohexane-carboxyl-CoA. The complex transformation of benzoyl-CoA is ascribed to at least two enzymes, benzoyl-CoA reductase (aromatic ring reducing) and cyclohex-1-enecarboxyl-CoA hydratase. The reduction of benzoyl-CoA to alicyclic compounds is catalyzed by extracts from cells grown anaerobically on either 2-aminobenzoate or benzoate at almost the same rate (10 to 15 nmol min-1 mg-1 of protein). In contrast, extracts from cells grown anaerobically on acetate or grown aerobically on benzoate or 2-aminobenzoate are inactive. This suggests a sequential induction of the enzymes.     

Anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) by a denitrifying bacterium

The anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) was studied in a denitrifying bacterium. Cells grown with 2-hydroxybenzoate were simultaneously adapted to degrade benzoate. Extract of these cells formed benzoate or benzoyl-CoA when incubated under reducing conditions with salicylate, MgATP, and coenzyme A, suggesting a degradation of 2-hydroxybenzoate via benzoate or benzoyl-CoA. This suggestion was supported by enzyme activity measurements. In extracts of 2-hydroxybenzoate-grown cells, the following enzyme activities were detected: two CoA ligases, one specific for 2-hydroxybenzoate, the other for benzoate, and two different enzyme activities catalyzing the reductive transformation of 2-hydroxybenzoyl-CoA. These findings suggest a degradation of salicylic acid by two new enzymes, 2-hydroxybenzoate-CoA ligase (AMP-forming) and 2-hydroxybenzoyl-CoA reductase (dehydroxylating), catalyzing (1) 2-hydroxybenzoate + MgATP + CoASH → 2-hydroxybenzoyl-CoA + MgAMP + PP_i (2) 2-hydroxybenzoyl-CoA + 2[H] → benzoyl-CoA + H₂O Benzoyl-CoA was dearomatized by reduction of the ring. This represents another case in which benzoyl-CoA is a central intermediate in anaerobic aromatic metabolism. Received: 1 February 1996 / Accepted: 24 February 1996     

Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl‐coenzyme A esters: organisms,strategies and key enzymes

Next to carbohydrates, aromatic compounds are the second most abundant class of natural organic molecules in living organic matter but also make up a significant proportion of fossil carbon sources. Only microorganisms are capable of fully mineralizing aromatic compounds. While aerobic microbes use well‐studied oxygenases for the activation and cleavage of aromatic rings, anaerobic bacteria follow completely different strategies to initiate catabolism. The key enzymes related to aromatic compound degradation in anaerobic bacteria are comprised of metal‐ and/or flavin‐containing cofactors, of which many use unprecedented radical mechanisms for C–H bond cleavage or dearomatization. Over the past decade, the increasing number of completed genomes has helped to reveal a large variety of anaerobic degradation pathways in Proteobacteria, Gram‐positive microbes and in one archaeon. This review aims to update our understanding of the occurrence of aromatic degradation capabilities in anaerobic microorganisms and serves to highlight characteristic enzymatic reactions involved in (i) the anoxic oxidation of alkyl side chains attached to aromatic rings, (ii) the carboxylation of aromatic rings and (iii) the reductive dearomatization of central arylcarboxyl‐coenzyme A intermediates. Depending on the redox potential of the electron acceptors used and the metabolic efficiency of the cell, different strategies may be employed for identical overall reactions.     

Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica—a Result of Promiscuous Enzymes and Regulators?

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO₂ as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.     

Aromatizing cyclohexa-1,5-diene-1-carbonyl-coenzyme A oxidase. Characterization and its role in anaerobic aromatic metabolism

Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive de-aromatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M(r) 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K(m) = 10 microm) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H(2)O. DCO was highly specific for 1,5-dienoyl-CoA (K(m) = 27 microm). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile de-aromatization/re-aromatization reactions catalyzed by BCR and DCO.     

Properties of 2-oxoglutarate:ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.     

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 E^o′ = −3 mV. 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.