Epoxy Coenzyme A Thioester Pathways for Degradation of AromaticCompounds

Wael Ismaila and Johannes Gescherb

Biotechnology Program, College of Graduate Studies, Arabian Gulf University, Manama, Kingdom of Bahrain,a and Karlsruhe Institute of Technology, Institute of AppliedBiosciences, Applied Biology, Karlsruhe, Germanyb

Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmen-tal pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routesthat enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability ofoxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In con-trast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel cata-bolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the establishedcatabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylace-tate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are boundby CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discussthe novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.

Aromatic compounds (natural and anthropogenic) comprisethe second largest group of organic carbon sources on earth,

next to carbohydrates (9, 12, 15, 24, 42, 83, 94). The literaturecomprises a large number of references reporting the ability ofbacteria (aerobic as well as anaerobic) and fungi to grow solelywith aromatic compounds, mainly of low molecular weight (4, 19,22, 23, 34, 45, 76, 77, 80, 91). The interest in studying the microbialmetabolism of aromatic compounds stems from its importantrole in the global carbon cycle and its direct impact on bioreme-diation of polluted ecosystems. Furthermore, many of the en-zymes involved in the degradation pathways are key catalysts inindustrial biotechnology (90). However, the microbial utilizationof aromatic compounds as a carbon and energy source is challeng-ing. Aromatics are relatively resistant to biomineralization due toan enhanced stability caused by the resonance energy of the aro-matic ring (150 kJ mol�1) (9). Therefore, microorganisms havedeveloped various catabolic strategies to cope with the inherentinertness of the aromatic compounds under oxic as well as anoxicconditions. The benzoate catabolic pathways are shown in Fig. 1.Under oxic conditions, microorganisms utilize oxygen as a cosub-strate to activate and subsequently cleave the aromatic ring (8, 11,39, 40, 47, 58, 74, 84). Substrate activation and ring cleavage arecatalyzed by oxygenases. The �-ketoadipate pathway is one of thewell-established aerobic catabolic routes and is named after acharacteristic intermediate, �-ketoadipate, that originates fromintradiol cleavage (40, 82) (Fig. 1). In contrast, under anoxic con-ditions, the aromatic compounds are first activated by formationof coenzyme A (CoA) thioesters. This is followed by an energy-consuming reduction of the ring and, eventually, a hydrolytic ringcleavage reaction (Fig. 1) (6, 7, 29, 41, 45, 50, 51).

Recently, a novel catabolic strategy for the aerobic degradationof aromatic compounds was elucidated. This new degradationroute differs significantly from the classical aromatic catabolicpathways (e.g., the �-ketoadipate pathway) (6, 40, 41, 43). It op-erates, for instance, in typical model organisms like Pseudomonasputida (67) and Escherichia coli (26) and was studied in detail for

the bacterial degradation of phenylacetate (PA) and benzoate andpartially investigated for the bacterial degradation of 2-aminoben-zoate (79). Obviously, microorganisms have evolved novel en-zymes to enable the adoption of unprecedented biochemical prin-ciples. In this review, we discuss the recently elucidated pathways,with a particular focus on the features that underpin their noveltyand ecological significance, and compare them to the well-estab-lished �-ketoadipate pathway.

THE PHENYLACETATE DEGRADATION PATHWAY

The here-described pathway is the only proven route for the bac-terial degradation of PA. It is, furthermore, necessary for the deg-radation of a multitude of other aromatic compounds which aremetabolized via PA, such as phenylalanine, phenylethylamine,and the environmental pollutants styrene and ethylbenzene (57)(Fig. 2). The biochemical characteristics of the novel PA degrada-tion strategy are aromatic ring activation via carbon-oxygen ep-oxide bond formation in combination with the production ofCoA-bound intermediates and an oxygen-independent (hydro-lytic) ring cleavage. Hence, modules from aerobic as well as anaer-obic metabolic pathways are combined in a single catabolic route(Fig. 2).

Activation of the substrate. Although the fungal metabolismof PA is well established and follows the aforementioned conven-tional strategy (63), the bacterial aerobic pathway remained enig-matic for more than 50 years (52). It was the discovery of specificPA-CoA ligases in PA-degrading bacteria 2 decades ago that initi-ated the belief in a nonconventional catabolic pathway (26, 67). Itis now textbook knowledge that the bacterial aerobic catabolism

Published ahead of print 11 May 2012

Address correspondence to Wael Ismail, waelame@agu.edu.bh.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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of phenylacetate starts with the transformation of PA to phenyl-acetyl-CoA (PACoA) (Fig. 2) (5, 26, 57, 59, 60, 61, 64, 89). Allfurther pathway intermediates are also CoA thioesters. Initiationof the aerobic degradation of aromatic compounds via CoA thio-esterification was reported for other aromatic acids as well (1, 16,30, 37, 48, 56, 68, 78, 92). The involvement of CoA thioesters in theaerobic degradation of aromatic compounds raises a questionabout the potential role played by CoA. Thioesterification of aro-matic acids usually takes place under anoxic conditions to activatethese compounds (7). The electron-withdrawing character of theCoA thioester facilitates the reduction of the aromatic ring, whichis the bottleneck of the anaerobic catabolic pathways (7, 29). Onecan assume a similar activation role for the CoA group under oxicconditions. In that sense, as discussed below, it has been shownthat CoA thioester formation mechanistically facilitates some ofthe reactions involved in PA aerobic catabolism. The significanceof CoA thioesters in the aerobic degradation pathways was re-cently reviewed (29).

Epoxidation of the aromatic ring. There has been a consensusthat the subsequent step in the PACoA degradation pathway is theintroduction of oxygen into the aromatic ring of PACoA. More-over, it has been generally accepted that the enzyme catalyzing theoxygenation reaction is a novel multicomponent complex en-coded by the paaABCDE genes. However, the nature of the oxy-

genation reaction has been controversial until recently. Ismail etal. (44) constructed single-knockout mutants by deleting someof the PA catabolic genes (paaFGHJZ) of E. coli K-12. They alsostudied the transformation of 13C-PA by cell suspensions ofthose mutants to elucidate the structure of accumulated path-way intermediates by 13C-nuclear magnetic resonance (13C-NMR) spectroscopy. They concluded that PACoA (the first inter-mediate) is transformed into a cis-dihydrodiol derivative (thesecond intermediate), thus suggesting a dioxygenase-catalyzedhydroxylation (Fig. 2). It is worth mentioning that the proposeddihydrodiol derivative could not be directly isolated and identi-fied. Nonetheless, its structure could be inferred from that of twodead-end products which were produced by one of the knockoutmutants (�paaG), namely, 2-hydroxyphenylacetate and 1,2-dihy-droxy-1,2-dihydrophenylacetyl lactone (Fig. 2). Recently, Teufelet al. (86) investigated the in vitro transformation of PACoA withpurified recombinant PaaABCDE. Structure elucidation of the re-action product performed using mass spectroscopy coupled with18O-labeling studies revealed the formation of ring 1,2-epoxyphe-nylacetyl-CoA (Fig. 2). Therefore, the putative PaaABCDE pro-tein complex was identified as a monooxygenase (ring 1,2-phenyl-acetyl-CoA epoxidase). The putative complex was fully active inthe absence of the PaaD subunit, which eluted separately duringpurification. Addition of recombinant PaaD to the in vitro assays

FIG 1 Overview of established degradation routes for benzoate that operate under oxic (A) or anoxic (B) conditions. (A) Aerobic degradation strategy based onhydroxylation of the aromatic ring and subsequent cleavage between hydroxyl groups (intradiol) or adjacent to the hydroxyl groups (extradiol). Intradiolcleavage of protocatechuate or catechol leads to �-ketoadipate as a characteristic intermediate. (B) Anaerobic degradation of aromatic compounds proceeds viabenzoyl-CoA, a characteristic intermediate. The dearomatization step is strictly endergonic. As an example, ATP usage as it occurs in Thauera aromatica is shown.Dashed arrows indicate that two or more enzymatic steps occur between the two depicted intermediates. Compound 1, benzoate; 2, 4-hydroxybenzoate; 3,protocatechuate; 4, 3-carboxy-cis,cis-muconate; 5, �-ketoadipate; 6, 1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate; 7, catechol; 8, cis,cis-muconate; 9, 2-hy-droxymuconic semialdehyde; 10, 2-hydroxy-4-carboxymuconic semialdehyde; 11, benzoyl-CoA; 12, 1,5-dienoyl-CoA; 13, 3-hydroxypimelyl-CoA.

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had no significant effect on the specific activity of the PaaABCEcomplex. The PaaD subunit might play a role in the maturation ofthe oxygenase and/or reductase component consisting of PaaACand/or PaaE, respectively (66). This assumption is based on thePaaD protein sequence similarity to SufT from the suf operon,which is involved in iron-sulfur cluster assembly (85, 86). More-over, the paaD gene is conserved among all bacteria possessing thepaa catabolic gene cluster for PA degradation, thus indicating thatit is essential. In line with these assumptions, studies on E. colisingle-knockout mutants of the paaABCDE genes showed thatPaaD is required for the catalysis in vivo (25). Therefore, it washypothesized that PaaD could be involved in the insertion of theiron-sulfur cluster in PaaE or the diiron center in PaaA (see below)(86).

The PaaABCE enzyme complex is the prototype of bacterialdiiron multicomponent monooxygenases (BMMs) acting on CoAthioesters (53). As mentioned above, the ring 1,2-PACoA epoxi-dase complex consists of four polypeptides encoded by thepaaABCE genes of the chromosomally located PA catabolic geneclusters of all PA-degrading bacteria (25). Structure-function in-vestigations of this novel enzyme appeared recently (35, 36). ThePaaAC subcomplex constitutes the oxygenase component, whichis involved in catalysis in analogy with the � (PaaA) and � (PaaC)subunits of BMMs. Although the overall architecture of the en-zyme is similar to that of other BMMs, the PaaAC subcomplexexhibits very low sequence identity to the oxygenase componentof other BMMs. The PaaAC subcomplex exists mainly as a(PaaAC)2 heterotetramer with an overall fold and core structure(B, C, E, F, G, and H �-helices) characteristic of the hydroxylase

component of monooxygenases. The B and C helices constitutethe interface of the PaaAC hydroxylase. The interface is hydro-phobic and has a large hydrophobic surface on the PaaA subunit.The two PaaAC heterodimers are arranged in an antiparallel fash-ion where the PaaA and PaaC subunits are anchored at the edgesof the interface via three salt bridges. The diiron and substratebinding sites are located in the PaaA subunit. In PaaA, the B, C, E,and F �-helices constitute a four-helix bundle-like fold with a longtunnel connecting the active center located in cavity 1 to the exte-rior of the protein. PaaC exhibits low sequence identity to PaaAand was proposed to play a structural role in the formation of theoxygenase subcomplex.

Electrons required for the catalysis are delivered from the re-ducing equivalent to the PaaAC subcomplex via the reductasecomponent PaaE (44, 63). The PaaE protein sequence shows sig-nificant similarity to members of the ferredoxin-NADP� reduc-tase (FNR) family. However, it exhibits an inverted domain order,i.e., an FNR-like N-terminal domain and a plant-type ferredoxinC-terminal domain. Altogether, these data indicate an iron-sulfurflavoprotein that transfers electrons from NADPH to the PaaA-located diiron center via flavin adenine dinucleotide (FAD) andan iron-sulfur cluster. Each PaaE component harbors one FADand one [2Fe-2S] binding domain (88).

Although the function of the small PaaB subunit (95 residues)is yet to be unraveled, it was shown to be essential for catalysis(36). Absence of PaaB resulted in a 100-fold decrease in productformation in vitro. Based on the stability of the PaaABC complexwith respect to ionic strength, it was hypothesized that PaaB playsa regulatory role in analogy with the � subunit of BMMs (36).

FIG 2 The aerobic phenylacetate (PA) catabolic pathway. Arrows pointing toward PA indicate some aromatic compounds that are degraded via PA. The thickarrow points toward a hypothetical intermediate. Dashed arrows indicate the dead-end products that were detected in studies on aerobic PA degradation. EB,ethylbenzene; STY, styrene; Phe, phenylalanine; CA, cinnamic acid; 2HPA, 2-hydroxyphenylacetate (dead-end product); HYP, hypothetical intermediate(cis-dihydrodiol derivative of PACoA); LACT, 1,2-dihydroxy-1,2-dihydrophenylacetyl lactone (dead-end product); HS-CoA, coenzyme A; PACoA, phenyl-acetyl-CoA; Ep-PACoA, ring-1,2-epoxy-PACoA; Oxepin-CoA, 2-oxepin-2 (3H)-ylideneacetyl-CoA; PaaK, PA-CoA ligase; PaaABCE, ring-1,2-PACoA epoxi-dase; PaaG, ring-1,2-epoxy-PACoA isomerase; PaaZ, oxepin-CoA hydrolase/3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase; PaaZ-ECHD, enoyl-CoA hydratase domain of the ring cleavage enzyme PaaZ; PaaZ-ALDH, aldehyde dehydrogenase domain of the ring cleavage enzyme PaaZ; PaaFGHJ, enzymescatalyzing �-oxidation-like reactions.

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Moreover, Fernández et al. and Ferrández et al. concluded thatPaaB is required for efficient synthesis of PaaC in vivo throughtranslational coupling (25, 26).

Despite similarities in overall protein topology, the PACoAepoxidase differs from members of the BMM family in many as-pects (36). For instance, the PaaA subunit is smaller (310 to 320amino acids) than the �-subunit of most BMMs (ca. 500 aminoacids) and the PaaE reductase component exhibits an inverteddomain order. Moreover, the Paa(AC)2 heterotetramers areformed via antiparallel interactions, in contrast to the parallelpacking observed in the ��� heterotrimers of BMM hydroxylasecomponents. In PaaA, cavity 1, which accommodates the acylmoiety of the substrate, is more hydrophilic than the correspond-ing hydrophobic region in the �-subunit of BMM hydroxylasecomponents. It is worth mentioning that residues lining cavity 1are determinants of substrate specificity and regiospecificity aswell as enantioselectivity. In this sense, it is argued that Phe-108 ofPaaA cavity 1 helps to position the PACoA substrate in the activecenter in such a way that the C1-C2 bond of the aromatic ring islocated above the diiron site, an orientation that is conductive toortho-hydroxylation. Differences can also be found in the diironbinding site, where one of the four glutamates commonly coordi-nating the diiron site in BMMs is replaced by an aspartate in PaaA.In addition, a salt bridge, the lysine bridge, is formed in closeproximity to the iron binding site between Lys-68 and the threeacidic residues Asp-126, Glu-72 (iron ligands), and Glu-49. Thelysine bridge is postulated to stabilize helices that constitute theferritin core, and it is lacking in other BMM members. Severalfurther features that characterize the members of the BMM familyare missing in the PACoA epoxidase as well. The hydroxylasecomponents of BMMs are known to possess hydrogen bond net-works connecting the active center with the surface of the proteinthat appear to mediate electron transfer from the reductase com-ponent to the diiron center at the hydroxylase component. Thisnetwork encloses residues from �-helices A, C, and F togetherwith the two histidines His-158 and His-75 that coordinate thediiron center. This hydrogen bond networking is lacking in thePaaA subunit of the PACoA epoxidase. Furthermore, �-helix Aand its homologues in the � and � subunits, which are crucial tothe oligomerization of the hydroxylase subunits, are also lackingin PaaAC. Two of the important catalytic residues (Thr-213 andAsn-214) that are conserved in BMM hydroxylase componentsare replaced in PaaA by Ala-129 and Ile-130. Altogether, theseunique features may be translated into different modes of oli-gomerization, catalysis, and electron transfer from the reductasePaaE to the diiron center.

In a recent study, Teufel et al. (88) showed that the PaaABCEepoxidase catalyzes an unprecedented conversion of the ring-1,2-epoxy-PACoA back to PACoA (the initial substrate) and water.This reaction proceeds when oxygen becomes limiting and it con-sumes NADPH (Fig. 2). When oxygen is introduced into the invitro assay, the reaction resumes its normal path and the epoxidasecatalyzes the transformation of PACoA to epoxy-PACoA. Accord-ingly, the PACoA epoxidase (PaaABCE) is a bifunctional enzyme(PACoA ring-1,2-epoxidase/ring-1,2-epoxy-PACoA deoxyge-nase). Deoxygenation of ring-1,2-epoxy-PACoA was proposed asa safety or detoxification mechanism that protects the cell fromthe hazardous epoxide when its further metabolism is impeded.

Isomerization and ring expansion. Two gene products, PaaGand PaaZ, of the paa-catabolic gene cluster were postulated to be

involved in ring cleavage. This suggestion was substantiated byevidence from in silico studies and knockout mutant investiga-tions which showed the formation of C8 dead-end products inpaaZ and paaG deletion mutants (44) (Fig. 2). It was shown re-cently that PaaG catalyzes the reversible transformation of thePACoA epoxide into an unusual unsaturated, oxygen-containing,seven-membered heterocyclic enol ether, namely, 2-oxepin-2(3H)-ylideneacetyl-CoA (oxepin-CoA) (86) (Fig. 2). Accordingly,PaaG functions as a ring 1,2-epoxyphenylacetyl-CoA isomerase(forming oxepin-CoA). In silico studies identified PaaG as a mem-ber of the crotonase superfamily, where it exhibits highest simi-larity to �3,�2-enoyl-CoA isomerases (26). Teufel et al. (86) pro-posed a mechanism for this reaction which involves cleavage ofthe two epoxy-C-O bonds and is facilitated by the electron-with-drawing character of the CoA thioester. This peculiar reaction isunprecedented in the aerobic degradation of aromatic com-pounds and is a further characteristic of the bacterial PA catabolicpathway. This leaves PaaZ as the only plausible candidate for thering fission.

Hydrolytic ring cleavage. Recently, Teufel et al. (87) presentedsubstantial evidence that the ultimate opening of the oxepin-CoAring is hydrolytic and catalyzed by PaaZ. This is another feature ofthe aerobic PA-catabolic pathway that is characteristic of theanaerobic degradation pathways. PaaZ is a bifunctional fusionprotein comprising two domains. The C-teminal domain (PaaZ-ECH) is a Hotdog-fold (R)-specific enoyl-CoA hydratase. It cata-lyzes the hydrolytic cleavage of the oxepin-CoA ring, resulting inan unstable open-chain aldehyde (Fig. 2). The latter is oxidized tothe corresponding carboxylic acid by the NADP�-dependent al-dehyde dehydrogenase domain that is localized to the N terminus(PaaZ-ALDH). In the absence of the aldehyde dehydrogenase ac-tivity, the highly reactive semialdehyde (product of ring cleavage)undergoes rapid intramolecular rearrangements (Knoevenagel-type condensation) to produce a more stable seven-memberedcarbon ring derivative which is a potent ring cleavage inhibitor(88) (Fig. 2). It was proposed that this dead-end product couldalso be the precursor for a number of secondary metabolites suchas antibiotics and unusual fatty acids. Interestingly, many bacterialack a PaaZ ortholog in their paa-catabolic gene cluster (75). In-stead, they possess an aldehyde dehydrogenase and the ring-cleav-ing enoyl-CoA hydratase is recruited from other genomic loca-tions that are unrelated to PA degradation. The mechanismproposed for the PaaZ-ECH-catalyzed cleavage of the oxepin-CoA ring involves an enolate anion intermediate whose formationis facilitated by the CoA thioester group. Certainly, this wouldjustify the need for CoA-bound intermediates. The final steps ofthe pathway are mediated by �-oxidation-like enzymes that suc-cessively transform the product of ring opening and aldehyde ox-idation to succinyl-CoA and 2 molecules of acetyl-CoA (Fig. 2).

The salvage enzymes. Interestingly, the PACoA pathway is en-dowed with several enzymes that protect the cell from metabolicbreakdown due to occasional accumulation of dead-end prod-ucts. The first enzyme is the hot dog-fold thioesterase PaaI, whichcatalyzes the release of CoA from PACoA and its hydroxylatedderivatives. Thus, PaaI avoids breakdown of intermediary metab-olism due to depletion of the CoA pool when the metabolism ofPACoA is blocked (44, 81). Furthermore, PaaI-catalyzed hydroly-sis of PACoA decreases the intracellular concentration of PACoAand favors the PaaABCE-catalyzed deoxygenation of epoxy-PACoA (88). Another thioesterase, PaaY, specifically removes

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CoA from 2-hydroxycyclohepta-1,4,6-triene-1-carboxyl-CoA(88). This compound is a dead-end product and ring cleavageinhibitor resulting from rearrangement of the ring cleavage prod-uct when the PaaZ-ALDH activity is impaired (Fig. 2). The PaaY-catalyzed removal of CoA avoids inhibition of ring cleavage andconsequently drives the pathway in the direction of epoxy-PACoAformation followed by ring opening. Therefore, the physiologicalrole of PaaY appears to be recovery of CoA from the dead-endproduct and stimulation of epoxy-PACoA formation (88). Theexpression of the paaI and paaY genes is induced in PA-growncells. The third salvage enzyme is the bifunctional PACoA epoxi-dase. This enzyme, as discussed above, catalyzes the transforma-tion (via deoxygenation) of its product epoxy-PACoA back toPACoA under oxygen-limiting conditions. This reaction relievesthe cell of epoxide stress (88). Another potential benefit from thisreaction is that it makes PACoA available for utilization via otherpathways in case its aerobic degradation is halted due to oxygenshortage. Facultative anaerobes, which likely face fluctuating ox-ygen tensions, can profit from this, because they can degradePACoA under anoxic conditions as well (29). It is worth mention-ing that the labile epoxy-PACoA rapidly and spontaneously de-cays to a nonutilizable dead-end product, 2-hydroxy-PA (Fig. 2).This explains why 2-hydroxy-PA was repeatedly detected in PAtransformation assays performed with cell suspensions and ex-tracts. It also justifies the need for a salvage reaction that circum-vents the rapid decay of epoxy-PACoA.

THE EPOXYBENZOYL-CoA PATHWAYEarly evidence for the epoxybenzoyl-CoA pathway. Already inthe 1970s, Clark and Buswell recognized that not all organismsseem to use the route of the �-ketoadipate pathway for the degra-dation of benzoate (10, 40). Their results showed that, in crudeextracts of certain thermophilic Bacillus species grown aerobicallyon benzoate, neither a protocatechuate- nor a catechol-dioxyge-nase was detectable (10). Similar observations were made laterwith the betaproteobacterium Azoarcus evansii and with a Geoba-cillus species (2, 46). Both organisms were not able to grow aero-bically with either protocatechuate or catechol as the carbonsource. Both compounds are intermediates of the aerobic cata-bolic routes. Hence, growth on those substrates would have beenexpected if the organisms use this strategy for their benzoate me-tabolism (Fig. 1) (1, 46, 65). More evidence for the existence of adifferent pathway arose after an aerobically induced benzoate-CoA ligase was found to catalyze the first step of the new pathwayin A. evansii. Meanwhile, most of the reactions and the catalyzingenzymes of this pathway were elucidated, and the rising number ofsequenced genomes reveals an increasing number of organismsthat harbor at least the necessary genes for the pathway (33).

Differences between the epoxybenzoyl-CoA pathway and the�-ketoadipate pathway are easily recognizable, since dissimilari-ties exist not only on the enzyme level, i.e., key enzymes are notrelated to enzymes of the established pathway, but also simply onthe level of the involved intermediates (31, 62, 72, 93) (Fig. 3).

FIG 3 The epoxybenzoyl-CoA pathway and the putative catalytic steps of the key reactions. Reactions catalyzed by BoxAB, BoxC, and BoxD are indicated.Compound 1, benzoate; 2, benzoyl-CoA; 3, epoxybenzoyl-CoA; 4, 3,4-dehydroadipyl-CoA-semialdehyde; 5, 3,4-dehydroadipyl-CoA; 6, 2,3-dehydroadipyl-CoA; 7, �-hydroxyadipyl-CoA; 8, �-ketoadipyl-CoA.

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Formation of epoxybenzoyl-CoA. It was first proposed byZaar et al. that a benzoyl-CoA dioxygenase would convert benzoyl-CoA to 2,3-dihydro-2,3-dihydroxybenzoyl-CoA using molecularoxygen as well as NADPH (91). Later, Rather et al. used the epox-ide trapping reagent N,N-diethyldithiocarbamate (DTC) in con-junction with mass spectrometry to identify the product as 2,3-epoxybenzoyl-CoA (72). DTC was in fact the key reagent, sincethe mass of the epoxide is identical to the mass of hydroxybenzoyl-CoA, which could result from water elimination from the for-merly proposed 2,3-dihydro-2,3-dihydroxybenzoyl-CoA. Thiscompound would be a likely artifact of an unstable dihydrodiol ina mass spectrometric experiment. The benzoyl-CoA, NADPH:ox-ygen oxidoreductase, is a two-component enzyme consisting ofthe reductase component BoxA and the oxidase component BoxB(where “Box” represents benzoyl-CoA oxidizing) (62, 92). BoxA isa dimeric iron-sulfur-flavoprotein. One BoxA monomer containstwo [4Fe-4S] clusters and FAD as cofactors. The [4Fe-4S] clustersshow extreme oxygen sensitivity, which becomes apparent by deg-radation to [3Fe-4S] clusters in the presence of even traceamounts of oxygen (71). This indicates a microoxic ecologicalniche of the organisms and is in line with hypotheses regarding theselective advantage of this pathway under low-oxygen conditions(see below). The oxidase BoxB is a diiron enzyme belonging to theclass I diiron protein family (see above). Consequently, BoxB con-tains two copies of the typical EXXH motifs (28, 55, 70). The twoglutamate and histidine residues are ligands of the binuclear ironcenter.

As 18O2-labeling experiments revealed, BoxB introduces oneoxygen atom from molecular oxygen into the substrate while theother is reduced to water (72) (Fig. 3). Interestingly, the activity ofthe oxygenase is impacted by the presence of BoxC (epoxyben-zoyl-CoA-converting enzyme). BoxC is necessary in vitro for acomplete conversion of benzoyl-CoA to downstream productsand accelerates the Vmax of the reaction to observed in vivo rates(31, 93).

BoxAB has few similarities to other ring-hydroxylating oxyge-nases (11). Instead of [2Fe-2S] clusters and an FAD in the reduc-tase component, BoxA contains two [4Fe-4S] clusters and anFAD. BoxB is a diiron enzyme instead of a protein containing aRieske [2Fe-2S] cluster and a mononuclear iron. Although BoxBis a class I diiron enzyme, it is still quite distinct from knownexamples such as stearyl-ACP �9 desaturase, ribonucleotide re-ductase, soluble methane monooxygenase, or the above-describedphenylacetyl-CoA epoxidase (27, 28, 49, 53, 54, 55). It is mono-meric and not part of a multimeric enzyme complex and showsfew sequence homologies to other members of the enzyme family.Still, catalytic mechanisms used by other diiron monooxygenasesas well as structural and Mössbauer spectroscopic data allowed theproposal of a mechanism of the BoxB-catalyzed reaction in whichthe resting state �-hydroxo Fe1III-(OH)2-Fe2III is reduced in aone-electron transfer step to the F1II-(OH)-Fe2III state (73) (Fig.3). Binding of benzoyl-CoA induces conformational changes inthe active center of BoxB, which might be the trigger for bind-ing of oxygen to the ferrous state Fe1 and the second one-electron transfer step. Similarities to methane monooxygenaselead to the postulation of an FeIV-(�-O)2-FeIV complex thatthereafter reacts in a putative radical mechanism with C2 andC3 of benzoyl-CoA. Eventually, epoxybenzoyl-CoA is formedby a simultaneous disintegration of the FeIV complex to anFeIII-O-FeIII complex (Fig. 3).

Hydrolytic ring cleavage. The other remarkable feature of theepoxybenzoyl-CoA pathway besides the epoxide formation is thehydrolytic instead of oxygenolytic ring cleavage. BoxC, the cata-lyzing enzyme, belongs to the enoyl-CoA hydratase/isomerase(crotonase) family of proteins (38). It is a homodimer with a na-tive molecular mass of 120 kDa (31) and was revealed via phylo-genetic analysis to be the most divergent member of the crotonasefamily identified so far (3). Bains and colleagues solved the BoxCcrystal structure recently (3). As expected, the active center con-tains two glutamate residues that can be superimposed on classicalmembers of the crotonase family (3, 38). These glutamates sup-port the formation of enolate anions. It was postulated that BoxCuses catalytic sequences known from other members of its proteinfamily for catalysis of the following reaction (31, 72):

2,3-epoxybenzoyl-CoA � 2 H2O

→ 3,4-dehydroadipyl-CoA-semialdehyde � HCOOH

Starting from epoxybenzoyl-CoA, it was postulated that BoxCopens the 2,3-epoxide or its oxepin tautomer at position C2 via aninitial addition of a hydroxyl group (Fig. 3). Further rearrange-ments would lead to a dialdehyde. Using a 2-ketocyclohexylcar-boxyl-CoA hydrolase-like activity, the upper formyl group is thenhydrolytically cleaved off, leading to the observed aldehyde withthe double bond in the 3,4 position (20, 21, 69) (Fig. 3). Interest-ingly, the C1 metabolic pathway in Burkholderia xenovorans isupregulated under conditions where the box gene cluster is in-duced (17, 18). This might be a response to the formate end prod-uct that is produced in the BoxC-catalyzed reaction. Formate de-hydrogenase could oxidize formate to CO2, which would lead tothe release of two reducing equivalents.

Further reactions of the epoxybenzoyl-CoA pathway. Thenext enzyme of the pathway was purified from A. evansii. BoxD isa NADP-specific aldehyde dehydrogenase which oxidizes the 3,4-dehydroadipyl-CoA semialdehyde to the corresponding acid (32)(compound 5; Fig. 3), thereby regenerating NADPH consumed inthe BoxAB-catalyzed reaction. Zaar et al. used cell extracts andprotein fractions of A. evansii to convert 13C-benzoyl-CoA intofurther intermediates of the pathway (Fig. 3) (92). NMR spectros-copy of the gained intermediates showed structures that seem toarise further downstream from 3,4-dehydroadipyl-CoA. The ob-served compounds would nicely fill the gap between this com-pound and the proposed product �-ketoadipyl-CoA. In detail,isomerization could lead from 3,4-dehydroadipyl-CoA to its 2,3isomer (compound 6; Fig. 3). This compound could be hydroxy-lated to �-hydroxyadipyl-CoA (compound 7; Fig. 3). An oxida-tion step would then lead to the so-far-undetected but likely in-termediate �-ketoadipyl-CoA (compound 8; Fig. 3).

Function of the epoxybenzoyl-CoA pathway as a new meta-bolic strategy. The occurrence of only CoA esters as intermediatesas well as the oxygen-independent ring cleavage are the two keyfeatures of the epoxybenzoyl-CoA pathway. Both features couldbe adaptations to low oxygen and benzoate concentrations in theecological niche of the organism as described below. It was shownby other groups that benzoate and catechol can leak out of the cellvia diffusion through the membrane (13, 17). Conversion of freeacids to CoA esters can trap the intermediates within a cell, toavoid loss of reaction products. Leakage of benzoyl-CoA and otherCoA-bound intermediates is greatly impaired compared to leak-age of free acids. This strategy was also found in E. coli, where afatty acid-CoA ligase is active in order to avoid drainage of the

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fatty acid out of the cell (14). Denef et al. were speculating that thehybrid pathway might be advantageous for an organism underconditions of low oxygen tensions, since here only half of theoxygen amount is needed for benzoate metabolism compared tothe �-ketoadipate pathway (17). The adaptation to low oxygentension could even go further, since, if the oxygen level dropsunder a certain threshold, the first intermediate of the benzoyl-CoA hybrid pathway is also the first intermediate of the anaerobicbenzoate metabolism. Therefore, a switch between aerobic andanaerobic degradation of benzoate would be fast and economic. Afurther hypothesis for a selective advantage of the epoxybenzoyl-CoA pathway over conventional pathways stems from the overallstoichiometries. The epoxybenzoyl-CoA pathway most probablyproceeds via the following equation:

benzoate � ATP � 2 CoA � O2 � 3H2O � NAD�

→ acetyl-CoA � succinyl-CoA � formic acid � AMP � PPi

� NADH � H�

NADH is likely formed during the conversion of �-hydroxy-adipyl-CoA to �-ketoadipyl-CoA. Comparing this to the overallstoichiometry of the �-ketoadipate pathway (benzoate � CoA �2O2 � H2O ¡ acetyl-CoA � succinate � CO2), it becomes ap-parent that the pathway not only needs half the amount of oxygenbut furthermore yields products like formate and NADH, whichcan be funneled into the respiratory chain via formate- andNADH-dehydrogenase, respectively. Hence, the strategy could beto spare the oxygenase cosubstrate oxygen in order to have moreof it as a terminal electron acceptor, which is necessary for energyproduction.

Occurrence of the epoxybenzoyl-CoA pathway among thebacteria. A database search revealed that 2% (60 genomes) of allbacterial genomes so far sequenced contain at least the genes forBoxB and BoxC. The same database search was conducted usingthe sequences of BenA and BenB from Pseudomonas putida, com-ponents of the benzoate 1,2-dioxygenase operating in the �-ketoadipate pathway. Here, 189 genomes were detected that con-tain genes annotated as, and therefore potentially encoding,benzoate 1,2-dioxygenase. Hence, a 3-fold-higher number wasidentified. Still, since the database contains a bias toward patho-genic organisms and a number of pathogens contain the benABgenes, it is very likely that the ratio of the environmental occur-rence of BoxBC to that of BenAB is far less than 1:3. Interestingly,20% of the genomes that contain BoxBC contain a copy of BenABin addition. Hence, a number of organisms might be able to useone pathway or the other, depending on certain environmentalconditions.

PERSPECTIVE

A number of examples in recent years have shown us that bacterialphysiology and biochemistry are still, after decades of research,full of surprises and novelties. The multitude of CO2 fixation path-ways discovered recently as well as the strategy for degradation ofaromatic compounds presented here can serve to illustrate this.The described pathways are characterized by the use of CoA thio-esters and hydrolytic ring cleavage and operate under oxic condi-tions. The PACoA pathway is widely distributed within the bacte-ria and is so far the only known pathway for degradation ofaromatics such as phenylalanine and phenylacetate for these or-ganisms. Still, there is no necessity to use the PACoA pathway,since within the fungi PA is degraded via conventional pathways.

Interestingly, the epoxybenzoyl-CoA pathway is also widely dis-tributed but is not the only bacterial strategy to cope with benzoateand substrates that are transformed to benzoate. Moreover, the�-ketoadipate pathway seems to be even more widely dispersed. Itshould be interesting to develop ideas and hypotheses that wouldexplain why in one case a given strategy might be superior enoughto fully push another strategy to the side or to prevent its develop-ment whereas in another case the same pathway might coexistwith another type of pathway.

ACKNOWLEDGMENTS

The research on the phenylacetate and benzoate degradation pathwayswas funded by Deutsche Forschungsgemeinschaft (DFG) and the SpanishMinistry of Education and Science.

We are indebted to Georg Fuchs, University of Freiburg, for unendingsupport and encouragement.

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