CHEMBIOCHEM MINIREVIEWS DOI: 10.1002/cbic.201402270
The Benzoyl-Coenzyme A Reductase and 2-HydroxyacylCoenzyme A Dehydratase Radical Enzyme Family Wolfgang Buckel,[b] Johannes W. Kung,[a] and Matthias Boll*[a]
Introduction Radical enzymes catalyse reactions via substrate-derived and product-related radical intermediates.[1] Radical enzymes are key players in universally important processes, such as anaerobic degradation of amino acids, alkanes or aromatic compounds, the conversion of pyruvate to acetyl-CoA, the synthesis of deoxyribonucleotides, Scheme 1. Proposed catalytic courses involving one-electron transfer steps. A) Benzoyl-CoA reductases: two ATPporphyrins and enzyme cofacdependent electron transfer events are involved per catalytic cycle B) (R)-2-hydroxyacyl-CoA dehydratases: the tors, as well as the maturation ATP-dependent electron transfer to the dehydratase component that initiates the redox-neutral dehydration is of transfer and ribosomal RNA. only catalytically required. The number of known/putative radical enzymes continues to increase, and many, especially from anaerobic bacteria, contain family” of phylogenetically related radical enzymes. Direct (metallo)organic cofactors such as vitamin B12, S-adenosylmethspectroscopic evidence for an allylic ketyl radical intermediate ionine (a glycyl-radical in the polypeptide chain), flavins or thihas recently been obtained for a HAD member,[6] and a number amine-diphosphate (for general reviews on radical enzymes of indirect observations argue for a related mechanism for see refs. [1a] and [2]). These cofactors expand the range of BCR.[7] enzyme catalysis at the risk of involving highly reactive intermediates. Here we describe a distinct radical enzyme family that contains only [4 Fe–4 S] clusters as prosthetic groups and yet catalGeneral Properties and Phylogeny of BCR/HAD yses, at first sight, totally unrelated reactions: the reduction of Family Members the aromatic ring of benzoyl-CoA to a cyclic dienoyl-CoA and Reaction catalysed by benzoyl-CoA reductases the elimination of water from 2-hydroxyacyl-CoA (Scheme 1).[3] In anaerobic bacteria most mono- and homocyclic aromatic Both reactions are mechanistically extremely difficult to achgrowth substrates are channelled into the central intermediate ieve, both use thioester substrates, both are oxygen-sensitive benzoyl-CoA, which is a substrate for dearomatising BCRs.[8] and contain active-site [4 Fe–4 S] clusters, both are ATP dependent and a radical mechanism involving ketyl intermediates These key enzymes catalyse the reduction of their substrates has been proposed for both (Scheme 1). Today it is evident by two electrons, thus yielding cyclohexa-1,5-diene-1-carboxylthat the 2-hydroxyacyl-CoA dehydratases (HADs)[4] and benzoCoA (1,5-dienoyl-CoA, Scheme 1 A), which is subjected to subsequent b-oxidation to acetyl-CoA. There are two completely yl-CoA reductases (BCRs)[5] are the prototypes of a “BCR/HAD different, non-related classes of BCRs: class I BCR is ATP-dependent and contains only [4 Fe–4 S] clusters as cofactors,[5, 9] [a] Dr. J. W. Kung, Prof. M. Boll whereas class II BCR is ATP-independent and contains an Institute of Biology II, University of Freiburg active-site tungstopterin, FeS clusters, FAD and selenocysSchnzlestrasse 1, 79104 Freiburg (Germany) E-mail: [email protected] teine.[9b, 10] In this review only the class I BCR related to HAD is [b] Prof. W. Buckel discussed; this is predominantly found in facultative anaerobes Laboratorium fr Mikrobiologie, Fachbereich Biologie such as denitrifying bacteria or bacteria with an anoxygenic Philipps-Universitt Marburg photosynthesis.[9b] Likely exceptions are the FeIII-reducing, Karl-von-Frisch-Strasse 8, 35043 Marburg (Germany) strictly anaerobic, hyperthermophilic Archaeon Ferroglobus Supporting information for this article is available on the WWW under placidus,[11] the naphthalene-degrading Deltaprotoebacterium http://dx.doi.org/10.1002/cbic.201402270. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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CHEMBIOCHEM MINIREVIEWS NaphS2[12] and the enrichment culture N47,[13] all of which are proposed to contain a class I BCR. In organic chemical synthesis, 1,4-dihydro addition to aromatic rings to yield non-conjugated cyclic dienes is a basic reaction known as Birch reduction.[14] It proceeds in alternating two single-electron transfer/protonation steps, with the first, rate-limiting electron transfer step yielding a radical anion. The one-electron reduction potential of non-substituted benzene to the corresponding radical anion is below 3 V.[15] For this reason the Birch reduction depends on solvated electrons as donors; these are the most potent reductants known in organic chemistry and are usually generated by dissolving alkali metals in liquid ammonia. Even in the case of the benzoic acid S-ethylthiol ester (an analogue of the biologically relevant benzoyl-CoA), the one-electron redox potential is with 1.9 V, far from the physiologically relevant range.[16] For this reason it was remarkable to discover enzymes that catalyse the reduction of benzoyl-CoA to 3,4-dihydrobenzoyl-CoA (1,5-dienoylCoA), similarly to the Birch reduction of benzoate to 1,4-dihydrobenzoate with elemental sodium in a mixture of methanol and liquid ammonia.[17] Notably, the Birch reduction can also yield the thermodynamically favoured conjugated 3,4-dihydrobenzoate by base-catalysed rearrangement.
www.chembiochem.org tonated at C3 to an allylic ketyl radical that is reoxidised by the enzyme to yield the product (E)-2-enoyl-CoA. Calculations indicated that formation of the enoxy radical lowered the pKa of the hydrogens at C3 from about 40 to 14.[21] This ketyl-radicalbased mechanism has been used as a guide for the characterisation of the dehydratase system by biochemical, spectroscopic and crystallographic methods.[3, 4]
Molecular architecture and phylogeny of class I BCRs and HADs BCRs and HADs have a common modular architecture with homologous subunits and similar cofactors (Figure 1).[4, 5, 20, 22] The electron-activation module is composed of two ATP binding site-containing subunits of the acetate and sugar kinase/heat shock cognate/actin (ASKHA) superfamily of phosphotransfer-
Reaction catalysed by (R)-2-hydroxyacyl-CoA dehydratase The syn elimination of water from 3-hydroxyacyl-CoA is wellstudied, and is a typical reaction of the b-oxidation of fatty acids catalysed by enoyl-CoA hydratases. The mechanism involves deprotonation of the relatively acidic proton at C2 (pKa < 10 of the enzyme-bound substrate),[18] with the leaving hydroxide group from C3 acting as the base.[19] However, during the syn dehydration of (R)-2-hydroxyacyl-CoA to (E)-2enoyl-CoA catalysed by HAD enzymes (Scheme 1 B), no such mechanism is possible, as the pKa of the hydrogen at C3 is around 40.[4] HADs play a role in the fermentation of various amino acids and (R)-2-hydroxy acids (e.g., d-lactate) in Firmicutes, Fusobacteria and the Archaeon Archaeoglobus fulgidus. Examples of HADs are (R)-lactyl-CoA dehydratase, (R)-2-hydroxyglutaryl-CoA dehydratase, (R)-phenyllactyl-CoA dehydratase and (R)-2-hydroxyisocaproyl-CoA dehydratase.[4, 20] During amino acid fermentation, the growth substrates are first converted into their 2-oxo acids by aminotransferases or dehydrogenases, and are then reduced to the corresponding (R)-2-hydroxy acids. Notably, the latter are always activated to their corresponding CoA esters by CoA-transferases prior to water elimination thus yielding the corresponding (E)-2-enoyl-CoA. The reductive elimination of a hydroxyl group adjacent to a ketone by one-electron donors such as Zn0, CrII or SmII is initiated by the reduction to a ketyl radical. Thereby the electrophilic carbonyl carbon suffers an “umpolung” to a nucleophile, which expels the hydroxyl group. Subsequent one-electron transfer to the transient enoxy radical affords the product.[3] In analogy, is has been postulated that dehydration of 2-hydroxyacyl-CoA involves the one-electron reduction of the thioester carbonyl to a ketyl radical, which eliminates the adjacent hydroxyl group. The resulting enoxy radical, however, is depro 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Architectures of prototypic members of the BCR/HAD family. Similar shades reflects amino acid similarities; similar sizes reflect similar subunits masses. All HgdC-like components are composed of two identical subunits, but the ATP-binding modules of the Azoarcus- (BzdPQ) and Thauera-type (BcrDA) BCRs differ in sequence and subunits mass.
ases;[23] the CoA-ester-binding module is formed of two additional subunits and harbours the active site. In the case of HAD, the two modules do not form a permanent complex,[24] whereas in at least some BCRs a stable complex is formed (Figure 1).[9a] The genes, architectures and cofactors of representative HADs (HgdCAB/LcdCAB/FldIBC/HadIBC)[4] and BCRs (BcrADBC/BadFGDE/BzdQPON)[9b] are listed in Table 1. The two subunits of the electron activation module contain a bridging [4 Fe–4 S] + 1/ + 2 cluster that is coordinated by two cysteines from each subunit.[25] This architecture resembles essentially the Fe-protein of nitrogenases,[26] although there is no marked amino acid sequence similarity. The [4 Fe–4 S] + 1/ + 2 cluster of BCR/HAD is the primary one-electron acceptor from the natural donor, generally a reduced ferredoxin.[27] In HAD the ATP-binding module is a homodimer (~ 28 kDa subunit) and is referred to as “activator” or “initiator” of the dehydratase.[25c] Depending on the type of HAD, the corresponding activator proteins are referred to as HgdC, HadI, LcdC or FldI (Table 1).[4] In BCR, the electron-activating module has a heterodimeric architecture with two similar but different subunits. Among BCRs, two types can be distinguished depending on the level ChemBioChem 2014, 15, 2188 – 2194
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Table 1. Molecular architecture and subunits of prototypic enzymes of the BCR/HAD family of radical enzymes. Organism
Enzyme
Electron-activating module[b] Subunits/GI Mass [kDa] Cofactors
T. aromatica
benzoyl-CoA reductase
BcrA:3724170 BcrD:3724171 BadF:503269128 BadG:503269127 BzdQ:45649069 BzdP:33326777 BzdQ:499555452 BzdP:499555453 HadI:54781340
Rhodopseudomonas palustris benzoyl-CoA reductase
48.4 30.2 47.7 29.2 32.5 28.0 32.4 27.9 28.4
Azoarcus sp. CIB
benzoyl-CoA reductase
Aromatoleum aromaticum
benzoyl-CoA reductase
C. difficile
(R)-2-hydroxyisocaproylCoA dehydratase (R)-2-hydroxyglutarylHgdC:433932 27.3 CoA dehydratase (R)-lactyl-CoA dehydratase LcdC:408407643 27.1
A. fermentans C. propionicum
Clostridium sporogenes Fusobacterium nucleatum
A. fulgidus
28.1
CoA-ester-binding module Subunits/GI Mass [kDa] Cofactors
1 [4 Fe–4 S][25a] BcrB:3724169 BcrC:3724168 1 [4 Fe–4 S][a] BadE:503269129 BadD:503269130 1 [4 Fe–4 S][a] BzdO:45649068 BzdN:45649067 1 [4 Fe–4 S][a] BzdO:499555454 BzdN:499555455 1 [4 Fe–4 S][52] HadB:54781342 HadC:54781343 1 [4 Fe–4 S][44] HgdA:296439332 HdgB:296439333 1 [4 Fe–4 S][a] LcdA:408407641 LcdB:408407642
48.8 43.7 49.5 44.0 50.5 43.4 49.9 43.4 46.3 42.4 53.9 42.0 47.4 41.8
1 [4 Fe–4 S][54]
46.2 43.1 ~ 50 ~ 42 ~ 26
(R)-phenyllactyl-CoA dehydratase (R)-2-hydroxyglutaryl-CoA dehydratase
FldI:16417588
HgdD: 19703551 ~ 27
1 [4 Fe–4 S][a]
(R)-phenyllactyl-CoA dehydratase
HgdC: 1485180
1 [4 Fe–4 S][a]
FldB:16417589 FldC:16417590 HgdA: 19703552 HgdB: 19703553 HgdC: 19703554 HgdA: 1485179 HgdB: 1485178
1 1 1 1 1 1 1 1 2
[4 Fe–4 S][25a] [4 Fe–4 S] [4 Fe–4 S][a] [4 Fe–4 S][a] [4 Fe–4 S][a] [4Fe4S][a] [4 Fe–4 S][a] [4Fe4S][a] [4 Fe–4 S][52]
1 [4 Fe–4 S] FMN, riboflavin[43] 2 [4 Fe–4 S] 0.5 FMN, 0.5 riboflavin[53] 2 [4 Fe–4 S][54] 1 [4 Fe–4 S][55] riboflavin 2 [4 Fe–4 S][a]
[a] Not experimentally verified.
of similarity between the two subunits (Figure 1): 1) Azoarcustype (BzdPQ) is more similar to HAD enzymes with two fairly similar subunits of 28–32 kDa, and 2) Thauera-type (BcrAD or BadFG) with different molecular masses (30 and > 45 kDa).[5, 9b] The CoA-ester-binding modules contain the active site and always comprise two different subunits (42–54 kDa). They generally harbour one or two [4 Fe–4 S] clusters; additionally, some (but not all) HADs contain varying numbers of FMN and riboflavin factors (Table 1, Figure 1).[4] The two active-site subunits show amino acid sequence identities of 26 % (BcrB/HgdA and BcrC/HgdB). Figure 2 shows a phylogenetic tree of the active site-containing BcrC/HgdB subunits of BCR/HAD family members with an amino acid sequence BLAST score higher than 100 (query sequence: BcrC from Thauera aromatica). Cluster I comprises all experimentally verified HgdB-like subunits from 2-hydroxyacyl-CoA dehydratases and many related sequences from Firmicutes, Fusobacteria and Spirochaetes that are proposed to be HAD enzymes. However, there are also HgdB-like sequences from Betaproteobacteria and Actinobacteria; these are not believed to ferment amino acids or 2-hydroxy acids. Cluster II comprises the experimentally verified BCR of the Thauera/Rhodopseudomonas-type (BcrC/BadB from Alpha- and Betaproteobacteria) and Azoarcus-type BCRs (BzdN from Betaproteobacteria, Ferroglobus placidus and a few Deltaproteobacteria). In addition, there are phylogenetic subclusters comprising BcrC-like putative gene products from bacteria that are not known to degrade aromatic compounds under anaerobic conditions, including Clostridia, Bacilli, Actinobacteria, aerobic Alpha- and Betaproteobacteria and methanogenic Archaea. In summary, there are numerous members of the BCR/HAD family with unknown functions, thus indicating that 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
the diversity of this enzyme family is still only poorly understood. A common mechanism of BCRs and HADs? Almost 20 years ago—several years before similarities of the encoding genes of BCRs and HADs were discovered—a common radical mechanism was proposed for both enzyme types.[3] It was based on the one-electron reduction of the thioester functionalities of benzoyl-CoA and the 2-hydroxyacyl-CoA substrates to ketyl intermediates (Scheme 1). In both cases, the redox potentials of this single electron transfer were considered to be far lower than those of any known natural electron donors. For this reason, electron transfer to the thioester has to be coupled the hydrolysis of ATP to ADP and Pi in both BCR and HAD. In the case of benzoyl-CoA, the formed ketyl can be stabilized by several resonance structures over the adjacent aromatic ring (Scheme 1). After protonation to a radical and the second ATP-dependent electron transfer to yield an anion, the second proton transfer yields the final dienoyl-CoA product. In total, two ATP molecules are hydrolysed for the transfer of two electrons.[28] In the case of HAD, umpolung from the nucleophilic thioester to the electrophilic ketyl facilitates the elimination of the adjacent 2-hydroxyl to an enoxy radical (Scheme 1). The pKa of the b-proton of the latter is more than 26 units higher than that of the thioester,[21, 29] and the formed allylic radical can readily be oxidized by the enzyme to the enoyl-CoA product. In summary, the whole process involves one-electron reduction of the thioester substrate by the enzyme and one-electron reChemBioChem 2014, 15, 2188 – 2194
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www.chembiochem.org 500 mV), electron transfer to the aromatic ring would be largely endergonic (DG’ +25 kJ mol 1). For this reason class I BCRs couple the endergonic electron transfer to stoichiometric ATP hydrolysis. All experimental data for the function of BCRs derive from studies with the enzyme from the denitrifying Betaproteobacterium T. aromatica.[9a] The catalytic cycle of this enzyme starts with an electron transfer from reduced ferredoxin (E0’ = 435 mV)[27a] to the [4 Fe–4 S] cluster of the BcrAD subunits 0’ (E 500 mV),[31] thereby reducing it to the +1 state (Scheme 2).[32] In this state, BCR hydrolyses ATP (to ADP + Pi),
Scheme 2. Catalytic cycle of BCR from T. aromatica. ATP/acyl-phosphate hydrolysis switches a [4 Fe–4 S] + 1 cluster from S = 1=2 to the EPR-detectable excited S = 7=2 state (inset). In the presence of substrate, electrons are transferred to the active site; in the absence of substrate, the enzyme falls back to the non-activated state, and the energy dissipates as heat.
Figure 2. Phylogenetic tree of the BCR/HAD family of radical enzymes. The tree was generated from the 283 amino acid sequences with highest similarities to BcrC (T. aromatica). Taxonomic classes are shown in different colours; known subunits of the BCR/HAD family are indicated by arrows. For tree generation, BLAST was used (http://blast.ncbi.nlm.nih.gov/Blast.cgi; expect threshold 10; word size 3; max matches in a query range 0; Matrix: BLOSUM62, Gap costs: existence, 11, extension: 1). Sequences with a BLAST score > 100 were used in Mega5.2 software (http://megasoftware.net/: neighbour joining; 1000 bootstrap replications; Poisson model; pairwise deletion of gaps/missing data). For details see Figure S1 in the Supporting Information.
duction of the enzyme by the transiently formed radical. Hence, the transferred electron is recycled after each turnover, and HAD needs only an initial ATP-dependent one-electron transfer from the activator module. The activated HAD can then catalyse more than 10 000 turnovers before an unspecific reoxidation of the enzyme requires a new round of ATP-dependent activation.[29] In contrast, BCRs depend on stoichiometric ATP hydrolysis for the two one-electron transfer steps to the aromatic substrate.[28]
Functions of BCRs ATP-dependent electron transfer The redox potential of the benzoyl-CoA/cyclohexa-1,5-dienoylCoA redox pair (E0’ = 622 mV) is among the most negative for a substrate/product redox couple in biology.[30] Even with a reduced ferredoxin (E’ under cellular conditions is around 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
with the transient formation of enzyme-derived acyl-phosphate intermediate.[33] Hydrolysis of ATP or acyl-phosphate induces a change of the spin-state of the [4 Fe–4 S] + 1 cluster from the usual S = 1=2 state to the rather unusual S = 7=2 high-spin state, possibly resulting in a marked lowering of its redox potential.[28] In the absence of benzoyl-CoA, the high-energy state switches back to the S = 1=2 ground state, and the energy dissipates as heat.[28] In the presence of benzoyl-CoA, electron transfer occurs from the reduced S = 7=2 cluster to the aromatic substrate via the two [4 Fe–4 S] cluster of the BcrBC subunits. Radical mechanism of substrate reduction Due to a lack of structural information, the mode of benzoylCoA binding in BCR is unknown. Similarities to the corresponding subunits of HAD enzymes (see below) suggest direct ligation to a [4 Fe–4 S] cluster at the carbonyl-oxygen. According to the proposed Birch-like mechanism, two possible transient substrate-based radical species are expected: the radical anion formed by one-electron reduction of benzoyl-CoA and the free radical obtained after protonation of the latter.[16] In single-turnover studies two transient isotropic electron paramagnetic resonance (EPR) signals were detected, with gvalues higher than those of typical organic radicals (g = 2.033 and 2.015), however a clear assignment to substrate-derived radical species was not possible.[34] Kinetic studies with a number of substrate analogues were in accordance with a Birchlike mechanism and suggested that the rate-limiting first electron transfer is probably assisted by a concerted proton transChemBioChem 2014, 15, 2188 – 2194
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CHEMBIOCHEM MINIREVIEWS fer.[35] The most convincing evidence for a Birch-like mechanism comes from two-dimensional NMR studies with labelled substrates.[7] When BCR from T. aromatica reacted with [D5]benzoyl-CoA in H2O, specific anti-dihydro-addition at C3 and C4 occurred. Surprisingly, exchange of a deuteron with the solvent was observed at C2; this can easily be explained with
Scheme 3. Stereochemical course and exchange reaction of BCR from T. aromatica. By using deuterated benzoyl-CoA (1), exchange of the deuteron at C2 is observed; this can be explained by the low pKa of the enoyl-CoA radical (4). Formation of the latter can be explained by protonation of the resonance structures (2 and 3) by a sterically flexible proton donor. Stereospecific anti-dihydro-addition at C3 and C4 leads to either 7 or 8 (which could not be differentiated by NMR).
a radical mechanism shown in Scheme 3. This mechanism allows reversible non-stereospecific protonation at C2, whereby proton addition from one side and proton abstraction from the other explains the observed exchange.[7] In contrast, protonation at C4 is most likely guided by the enzyme with a specific proton donor. BCRs can degrade numerous substrate analogues that are readily reduced by two electrons. In the case of 2-hydroxyand 2-amino-benzoyl-CoA the formed 1,5-dienoyl-products tautomerise/hydrolyse to the 6-oxocyclohex-1-ene-1-carboxylCoA, a common intermediate of the benzoyl-CoA degradation pathway.[9b] In the case of 3-hydroxy-,[36] 3-methyl-[37] and 4methyl-benzoyl-CoA[38] the further catabolism of the dienoylCoA analogues requires a modified benzoyl-CoA degradation pathway. With 3-chlorobenzoyl-CoA as substrate, intrinsic BCR dehalogenase activity was recently identified: the chlorinated substrate is first reduced to the corresponding 3-chloro-1,5-dienoyl-CoA, which then eliminates HCl to yield the rearomatised benzoyl-CoA.[39] A class I BCR-like reaction was also identified in the anaerobic degradation of naphthalene in sulfate-reducing bacteria; here the intermediate 5,6,7,8-tetrahydro-2-naphthoylCoA was reduced to a hexahydronaphthoyl-CoA product in an ATP-dependent manner.[40] 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org Function of HADs ATP-dependent electron transfer Treatment of 2-hydroxyisocaproyl-CoA dehydratase (HadBC) with deazaflavin, EDTA and light (E0’ 650 mV)[41] generated an enzyme with up to 50 % of the activity achieved with the reduced activator HadI in the presence of ATP and Mg2 + (J. Kim and W.B., unpublished results). Hence, for one-electron reduction of the thioester carbonyl to a ketyl radical, a redox potential of about 800 mV could be feasible. As the activator is completely reduced with ferredoxin (E0’ 405 mV),[27b] the redox potential of the activator should be about 300 mV. Thus, hydrolysis of ATP must overcome a difference of ~ 500 mV. By taking DG’ = 50 kJ mol 1 for ATP hydrolysis under physiological conditions (250 mV for the transfer of one electron), two ATP would be sufficient to reach the extremely low potential of 800 mV. The mechanism by which hydrolysis of ATP drives electron transfer from the activator to the dehydratase is still elusive. The four characterised activators share 60–70 % sequence identity and are extremely oxygen-sensitive homodimeric proteins with one [4 Fe–4 S] cluster between the two subunits. The crystal structure of HgdC (3.0 resolution) indeed revealed that the [4 Fe–4 S]2 + cluster is bound at the centre of the activator, coordinated by four cysteines (two from each subunit; Figure 1).[25c] The most remarkable feature of HgdC is that helix 5 of each subunit points towards the cluster, thereby forming a helix-cluster-helix angle of about 1058 (see the similar structure of HadI, Figure 3). The diamagnetic [4 Fe–4 S]2 + cluster is
Figure 3. Views of the activator HadI from C. difficile with bound ADP showing the two subunits (green, blue), [4 Fe–4 S] cluster (yellow (S) and brown (Fe) balls) and ADP (red sticks). Adapted from ref. [13].
easily reduced to [4 Fe–4 S] + with dithionite, TiIII citrate and reduced ferredoxin2 or flavodoxin2 .[27b, 42] EPR spectroscopy revealed spin S = 3=2 for the [4 Fe–4 S] + cluster (g = 4–6) rather than the usual spin (S = 1=2 ), probably due to distortion of the cluster between the two subunits.[43] With excess TiIII citrate, 50 % reduction of HgdC with two electrons to the inactive allferrous state [4 Fe–4 S]0 was achieved.[44] The only other biological example of an all-ferrous cluster is the iron protein of nitrogenase, which has similar properties.[45] ChemBioChem 2014, 15, 2188 – 2194
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CHEMBIOCHEM MINIREVIEWS It has been proposed that upon binding of two ATP molecules to the activator and complex formation with the dehydratase, the angle opens to 1808 and shifts the reduced cluster towards the b-cluster of the dehydratase thus facilitating the one-electron transfer.[4] This hypothesis stems from the crystal structure of the phylogenetically unrelated but functionally similar iron protein of nitrogrenase, which shows almost the same architecture (helix-cluster-helix angle of 1508).[46] In complex with the MoFe protein of nitrogenase stabilized with ADP–AlF3, the angle opened to almost 1808. Although attempts to generate such complexes with HgdC from Acidaminococcus fermentans and HgdAB from Clostridium symbiosum (M. Hetzel and W.B., unpublished results) as well as with HadI and HadBC from Clostridium difficile[24] were successful, diffracting crystals could not be obtained.[25b] Recently crystal structures of reduced HadI in the presence of ADP (1.9 resolution; Figure 3), ADPNP (1.6 ) and ADP-AlF3 (3.0 ) were obtained.[25b] Unexpectedly, the conformational changes induced by the reduced cluster and ligands were very small (and only local), compared to the structure of oxidized HgdC containing ADP. However, taking into account that efficient ATP hydrolysis requires not only the reduced cluster but also the dehydratase,[47] the predicted large conformational change will become visible only in the elusive activator–dehydratase complexes.
Radical mechanism of water elimination The crystal structure of 2-hydroxyisocaproyl-CoA dehydratase (HadBC) from C. difficile immediately gave clues to the mechanism (Figure 4). Each subunit contains one [4 Fe–4 S] cluster coordinated by three conserved cysteines. In the HadB subunit (here referred to as the a-subunit) the ligand of the fourth iron of the a-cluster is a hydroxyl group, whereas in the b-cluster of the b-subunit (subunit HadC) a sulfhydryl group is at this position. Such a ligand has never been detected in other [4 Fe–4 S]
Figure 4. Structure of (R)-2-hydroxyisocaproyl-CoA dehydratase. The a-subunit is coloured according to its three domains. The b-subunit is coloured according to secondary structure: a-helices (blue), b-sheets (orange) and loops (green). The [4 Fe–4 S] clusters with terminal ligands are shown as spheres: sulfur (yellow-green), iron (orange) and oxygen (red). Adapted from ref. [21].
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www.chembiochem.org clusters. Thus the b-cluster can only act as electron reservoir because there is no possible access for a substrate. In contrast, the a-cluster has access to the protein surface by a channel through which the substrate can approach. Soaking experiments with (R)-2-hydroxyisocaproyl-CoA indeed revealed that the thioester carbonyl replaces the hydroxyl group at the acluster and is hydrogen bonded to a conserved Glu55–Ser37 dyad. The putative mechanism starts with binding of the reduced activator loaded with two ATP molecules at one side of the dehydratase; the closest distance of the activator cluster to the b-cluster is about 12 . Hydrolysis of ATP “shoots” the electron into the b-cluster, which stores the electron until (R)-2-hydroxyisocaproyl-CoA binds to the a-cluster thus replacing the hydroxide at the fourth iron. The removal of several water molecules by the substrate induces an 11 electron transfer from the b- to the a-cluster. Next an inner shell electron transfer to the thioester carbonyl generates the ketyl radical, which (a less efficient Lewis base) leaves the iron and is replaced by the hydroxyl group of the substrate. This “ligand swapping” facilitates the elimination of the hydroxide, which acts as a base to remove the now acidic proton at C3.[21] The stereochemistry of this elimination is consistent with the overall syn geometry of this process.[4] The resulting allylic ketyl radical, which has been identified by EPR spectroscopy,[6] again binds to the fourth iron of the a-cluster and returns its electron, which is used for the next turnover or is stored in the b-cluster.
Outlook Over the past decade, substantial progress in the knowledge of the function of the BCR/HAD family has been achieved. Especially in the case of HAD enzymes, direct spectroscopic evidence of a substrate-based radical and the first crystal structure have largely contributed to a better understanding of the function of this enzyme family. In the case of BCRs, there is still a great need for research to provide evidence of the proposed Birch-like mechanism. For the BCR/HAD family there are perspectives for applied approaches. For example, glutaconic acid production was achieved by introducing six genes from glutamate-fermenting bacteria into Escherichia coli (2-hydroxyglutaryl-CoA dehydratase was the key enzyme).[48] E. coli was converted into a propionate producer by using the genes for lactyl-CoA dehydratase and other enzymes from Clostridium propionicum.[49] With the same promiscuous enzymes adipate can be formed, from which Nylon 6,6 can be produced.[50] Despite its great utility, chemical Birch reduction of aromatic rings has several undesirable attributes, including the handling of alkali metals, cryogenic reaction conditions and the toxicity of the ammonia. For this reasons, there is a high demand for alternatives.[51] The high stereospecificity and the facile conversion of numerous substrate analogues make enzymatic Birch reduction by BCRs an attractive alternative to accomplish stereospecific dihydro additions to aromatic CoA ester substrates. ChemBioChem 2014, 15, 2188 – 2194
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Acknowledgements W.B. acknowledges support from the German Research Foundation (DFG), the Max-Planck Society and the Loewe Center of Synthetic Microbiology (SYNMIKRO). Keywords: ATP-dependent electron transfer · enzymes · iron– sulfur cluster · ketyl radical · radical reactions [1] a) W. Buckel, Angew. Chem. Int. Ed. 2009, 48, 6779 – 6787; Angew. Chem. 2009, 121, 6911 – 6920; b) P. A. Frey, Chem. Rec. 2001, 1, 277 – 289. [2] a) W. Buckel, B. T. Golding, Annu. Rev. Microbiol. 2006, 60, 27 – 49; b) P. A. Frey, A. D. Hegeman, G. H. Reed, Chem. Rev. 2006, 106, 3302 – 3316; c) P. A. Frey, A. D. Hegeman, F. J. Ruzicka, Crit. Rev. Biochem. Mol. Biol. 2008, 43, 63 – 88. [3] W. Buckel, R. Keese, Angew. Chem. Int. Ed. Engl. 1995, 34, 1502 – 1506; Angew. Chem. 1995, 107, 1595 – 1598. [4] W. Buckel, J. Zhang, P. Friedrich, A. Parthasarathy, H. Li, I. Djurdjevic, H. Dobbek, B. M. Martins, Biochim. Biophys. Acta 2012, 1824, 1278 – 1290. [5] M. Boll, J. Mol. Microbiol. Biotechnol. 2005, 10, 132 – 142. [6] J. Kim, D. J. Darley, W. Buckel, A. J. Pierik, Nature 2008, 452, 239 – 242. [7] B. Thiele, O. Rieder, B. T. Golding, M. Mller, M. Boll, J. Am. Chem. Soc. 2008, 130, 14050 – 14051. [8] G. Fuchs, M. Boll, J. Heider, Nat. Rev. Microbiol. 2011, 9, 803 – 816. [9] a) M. Boll, G. Fuchs, Eur. J. Biochem. 1995, 234, 921 – 933; b) M. Boll, C. Lçffler, B. E. Morris, J. W. Kung, Environ. Microbiol. 2014, 16, 612 – 627. [10] a) J. W. Kung, C. Lçffler, K. Dçrner, D. Heintz, S. Gallien, A. Van Dorsselaer, T. Friedrich, M. Boll, Proc. Natl. Acad. Sci. USA 2009, 106, 17687 – 17692; b) C. Lçffler, K. Kuntze, J. R. Vazquez, A. Rugor, J. W. Kung, A. Bçttcher, M. Boll, Environ. Microbiol. 2011, 13, 696 – 709. [11] D. E. Holmes, C. Risso, J. A. Smith, D. R. Lovley, ISME J. 2012, 6, 146 – 157. [12] R. J. DiDonato, Jr., N. D. Young, J. E. Butler, K.-J. Chin, K. K. Hixson, P. Mouser, M. S. Lipton, R. DeBoy, B. A. Meth, PLoS One 2010, 5, e14072. [13] F. Bergmann, D. Selesi, T. Weinmaier, P. Tischler, T. Rattei, R. U. Meckenstock, Environ. Microbiol. 2011, 13, 1125 – 1137. [14] a) A. J. Birch, J. Chem. Soc. 1944, 430 – 436; b) H. E. Zimmerman, Acc. Chem. Res. 2012, 45, 164 – 170. [15] J. Mortensen, J. Heinze, Angew. Chem. Int. Ed. Engl. 1984, 23, 84 – 85; Angew. Chem. 1984, 96, 64 – 65. [16] M. Boll, D. Laempe, W. Eisenreich, A. Bacher, T. Mittelberger, J. Heinze, G. Fuchs, J. Biol. Chem. 2000, 275, 21889 – 21895. [17] H. Plieninger, G. Ege, Angew. Chem. 1958, 70, 505 – 506. [18] S. Ghisla, C. Thorpe, Eur. J. Biochem. 2004, 271, 494 – 508. [19] B. J. Bahnson, V. E. Anderson, G. A. Petsko, Biochemistry 2002, 41, 2621 – 2629. [20] W. Buckel, B. M. Martins, A. Messerschmidt, B. T. Golding, Biol. Chem. 2005, 386, 951 – 959. [21] D. M. Smith, W. Buckel, H. Zipse, Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870; Angew. Chem. 2003, 115, 1911 – 1915. [22] M. Boll, Biochim. Biophys. Acta Bioenerg. 2005, 1707, 34 – 50. [23] K. A. Buss, D. R. Cooper, C. Ingram-Smith, J. G. Ferry, D. A. Sanders, M. S. Hasson, J. Bacteriol. 2001, 183, 680 – 686. [24] J. Kim, A. J. Pierik, W. Buckel, ChemPhysChem 2010, 11, 1307 – 1312. [25] a) M. Boll, G. Fuchs, C. Meier, A. Trautwein, D. J. Lowe, J. Biol. Chem. 2000, 275, 31857 – 31868; b) S. H. Knauer, W. Buckel, H. Dobbek, Bio-
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chemistry 2012, 51, 6609 – 6622; c) K. P. Locher, M. Hans, A. P. Yeh, B. Schmid, W. Buckel, D. C. Rees, J. Mol. Biol. 2001, 307, 297 – 308. W. Buckel, M. Hetzel, J. Kim, Curr. Opin. Chem. Biol. 2004, 8, 462 – 467. a) M. Boll, G. Fuchs, Eur. J. Biochem. 1998, 251, 946 – 954; b) W. Thamer, I. Cirpus, M. Hans, A. J. Pierik, T. Selmer, E. Bill, D. Linder, W. Buckel, Arch. Microbiol. 2003, 179, 197 – 204. M. Boll, S. S. P. Albracht, G. Fuchs, Eur. J. Biochem. 1997, 244, 840 – 851. J. Kim, D. Darley, W. Buckel, FEBS J. 2005, 272, 550 – 561. J. W. Kung, S. Baumann, M. von Bergen, M. Mller, P.-L. Hagedoorn, W. R. Hagen, M. Boll, J. Am. Chem. Soc. 2010, 132, 9850 – 9856. M. Boll, G. Fuchs, C. Meier, A. Trautwein, A. El Kasmi, S. W. Ragsdale, G. Buchanan, D. J. Lowe, J. Biol. Chem. 2001, 276, 47853 – 47862. H. Mçbitz, T. Friedrich, M. Boll, Biochemistry 2004, 43, 1376 – 1385. M. Unciuleac, M. Boll, Proc. Natl. Acad. Sci. USA 2001, 98, 13619 – 13624. M. Boll, G. Fuchs, D. J. Lowe, Biochemistry 2001, 40, 7612 – 7620. H. Mçbitz, M. Boll, Biochemistry 2002, 41, 1752 – 1758. D. Laempe, M. Jahn, K. Breese, H. Schgger, G. Fuchs, J. Bacteriol. 2001, 183, 968 – 979. J. F. Jurez, M. T. Zamarro, C. Eberlein, M. Boll, M. Carmona, E. Daz, Environ. Microbiol. 2013, 15, 148 – 166. S. Lahme, C. Eberlein, R. Jarling, M. Kube, M. Boll, H. Wilkes, R. Reinhardt, R. Rabus, Environ. Microbiol. 2012, 14, 1118 – 1132. K. Kuntze, P. Kiefer, S. Baumann, J. Seifert, M. von Bergen, J. A. Vorholt, M. Boll, Mol. Microbiol. 2011, 82, 758 – 769. C. Eberlein, J. Johannes, H. Mouttaki, M. Sadeghi, B. T. Golding, M. Boll, R. U. Meckenstock, Environ. Microbiol. 2013, 15, 1832 – 1841. G. Blankenhorn, Eur. J. Biochem. 1976, 67, 67 – 80. M. Hans, E. Bill, I. Cirpus, A. J. Pierik, M. Hetzel, D. Alber, W. Buckel, Biochemistry 2002, 41, 5873 – 5882. M. Hans, W. Buckel, E. Bill, Eur. J. Biochem. 2000, 267, 7082 – 7093. M. Hans, W. Buckel, E. Bill, J. Biol. Inorg. Chem. 2008, 13, 563 – 574. H. C. Angove, S. J. Yoo, B. K. Burgess, E. Mnck, J. Am. Chem. Soc. 1997, 119, 8730 – 8731. H. Schindelin, C. Kisker, J. L. Schlessman, J. B. Howard, D. C. Rees, Nature 1997, 387, 370 – 376. J. Kim, Y. Lu, W. Buckel, Comptes Rendus Chimie 2007, 10, 742 – 747. I. Djurdjevic, O. Zelder, W. Buckel, Appl. Environ. Microbiol. 2011, 77, 320 – 322. V. Kandasamy, H. Vaidyanathan, I. Djurdjevic, E. Jayamani, K. B. Ramachandran, W. Buckel, G. Jayaraman, S. Ramalingam, Appl. Microbiol. Biotechnol. 2013, 97, 1191 – 1200. A. Parthasarathy, A. J. Pierik, J. Kahnt, O. Zelder, W. Buckel, Biochemistry 2011, 50, 3540 – 3550. a) M. J. Costanzo, M. N. Patel, K. A. Petersen, P. F. Vogt, Tetrahedron Lett. 2009, 50, 5463 – 5466; b) P. Nandi, J. L. Dye, J. E. Jackson, J. Org. Chem. 2009, 74, 5790 – 5792. S. H. Knauer, W. Buckel, H. Dobbek, J. Am. Chem. Soc. 2011, 133, 4342 – 4347. R. D. Kuchta, G. R. Hanson, B. Holmquist, R. H. Abeles, Biochemistry 1986, 25, 7301 – 7307. S. Dickert, A. J. Pierik, W. Buckel, Mol. Microbiol. 2002, 44, 49 – 60. A. Tamannaei, Diploma Thesis, Philipps-Universitt Marburg (Germany), 2003.
Received: May 28, 2014 Published online on September 9, 2014
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The Benzoyl-Coenzyme A Reductase and 2-HydroxyacylCoenzyme A Dehydratase Radical Enzyme Family Wolfgang Buckel,[b] Johannes W. Kung,[a] and Matthias Boll*[a]
Introduction Radical enzymes catalyse reactions via substrate-derived and product-related radical intermediates.[1] Radical enzymes are key players in universally important processes, such as anaerobic degradation of amino acids, alkanes or aromatic compounds, the conversion of pyruvate to acetyl-CoA, the synthesis of deoxyribonucleotides, Scheme 1. Proposed catalytic courses involving one-electron transfer steps. A) Benzoyl-CoA reductases: two ATPporphyrins and enzyme cofacdependent electron transfer events are involved per catalytic cycle B) (R)-2-hydroxyacyl-CoA dehydratases: the tors, as well as the maturation ATP-dependent electron transfer to the dehydratase component that initiates the redox-neutral dehydration is of transfer and ribosomal RNA. only catalytically required. The number of known/putative radical enzymes continues to increase, and many, especially from anaerobic bacteria, contain family” of phylogenetically related radical enzymes. Direct (metallo)organic cofactors such as vitamin B12, S-adenosylmethspectroscopic evidence for an allylic ketyl radical intermediate ionine (a glycyl-radical in the polypeptide chain), flavins or thihas recently been obtained for a HAD member,[6] and a number amine-diphosphate (for general reviews on radical enzymes of indirect observations argue for a related mechanism for see refs. [1a] and [2]). These cofactors expand the range of BCR.[7] enzyme catalysis at the risk of involving highly reactive intermediates. Here we describe a distinct radical enzyme family that contains only [4 Fe–4 S] clusters as prosthetic groups and yet catalGeneral Properties and Phylogeny of BCR/HAD yses, at first sight, totally unrelated reactions: the reduction of Family Members the aromatic ring of benzoyl-CoA to a cyclic dienoyl-CoA and Reaction catalysed by benzoyl-CoA reductases the elimination of water from 2-hydroxyacyl-CoA (Scheme 1).[3] In anaerobic bacteria most mono- and homocyclic aromatic Both reactions are mechanistically extremely difficult to achgrowth substrates are channelled into the central intermediate ieve, both use thioester substrates, both are oxygen-sensitive benzoyl-CoA, which is a substrate for dearomatising BCRs.[8] and contain active-site [4 Fe–4 S] clusters, both are ATP dependent and a radical mechanism involving ketyl intermediates These key enzymes catalyse the reduction of their substrates has been proposed for both (Scheme 1). Today it is evident by two electrons, thus yielding cyclohexa-1,5-diene-1-carboxylthat the 2-hydroxyacyl-CoA dehydratases (HADs)[4] and benzoCoA (1,5-dienoyl-CoA, Scheme 1 A), which is subjected to subsequent b-oxidation to acetyl-CoA. There are two completely yl-CoA reductases (BCRs)[5] are the prototypes of a “BCR/HAD different, non-related classes of BCRs: class I BCR is ATP-dependent and contains only [4 Fe–4 S] clusters as cofactors,[5, 9] [a] Dr. J. W. Kung, Prof. M. Boll whereas class II BCR is ATP-independent and contains an Institute of Biology II, University of Freiburg active-site tungstopterin, FeS clusters, FAD and selenocysSchnzlestrasse 1, 79104 Freiburg (Germany) E-mail: [email protected] teine.[9b, 10] In this review only the class I BCR related to HAD is [b] Prof. W. Buckel discussed; this is predominantly found in facultative anaerobes Laboratorium fr Mikrobiologie, Fachbereich Biologie such as denitrifying bacteria or bacteria with an anoxygenic Philipps-Universitt Marburg photosynthesis.[9b] Likely exceptions are the FeIII-reducing, Karl-von-Frisch-Strasse 8, 35043 Marburg (Germany) strictly anaerobic, hyperthermophilic Archaeon Ferroglobus Supporting information for this article is available on the WWW under placidus,[11] the naphthalene-degrading Deltaprotoebacterium http://dx.doi.org/10.1002/cbic.201402270. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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CHEMBIOCHEM MINIREVIEWS NaphS2[12] and the enrichment culture N47,[13] all of which are proposed to contain a class I BCR. In organic chemical synthesis, 1,4-dihydro addition to aromatic rings to yield non-conjugated cyclic dienes is a basic reaction known as Birch reduction.[14] It proceeds in alternating two single-electron transfer/protonation steps, with the first, rate-limiting electron transfer step yielding a radical anion. The one-electron reduction potential of non-substituted benzene to the corresponding radical anion is below 3 V.[15] For this reason the Birch reduction depends on solvated electrons as donors; these are the most potent reductants known in organic chemistry and are usually generated by dissolving alkali metals in liquid ammonia. Even in the case of the benzoic acid S-ethylthiol ester (an analogue of the biologically relevant benzoyl-CoA), the one-electron redox potential is with 1.9 V, far from the physiologically relevant range.[16] For this reason it was remarkable to discover enzymes that catalyse the reduction of benzoyl-CoA to 3,4-dihydrobenzoyl-CoA (1,5-dienoylCoA), similarly to the Birch reduction of benzoate to 1,4-dihydrobenzoate with elemental sodium in a mixture of methanol and liquid ammonia.[17] Notably, the Birch reduction can also yield the thermodynamically favoured conjugated 3,4-dihydrobenzoate by base-catalysed rearrangement.
www.chembiochem.org tonated at C3 to an allylic ketyl radical that is reoxidised by the enzyme to yield the product (E)-2-enoyl-CoA. Calculations indicated that formation of the enoxy radical lowered the pKa of the hydrogens at C3 from about 40 to 14.[21] This ketyl-radicalbased mechanism has been used as a guide for the characterisation of the dehydratase system by biochemical, spectroscopic and crystallographic methods.[3, 4]
Molecular architecture and phylogeny of class I BCRs and HADs BCRs and HADs have a common modular architecture with homologous subunits and similar cofactors (Figure 1).[4, 5, 20, 22] The electron-activation module is composed of two ATP binding site-containing subunits of the acetate and sugar kinase/heat shock cognate/actin (ASKHA) superfamily of phosphotransfer-
Reaction catalysed by (R)-2-hydroxyacyl-CoA dehydratase The syn elimination of water from 3-hydroxyacyl-CoA is wellstudied, and is a typical reaction of the b-oxidation of fatty acids catalysed by enoyl-CoA hydratases. The mechanism involves deprotonation of the relatively acidic proton at C2 (pKa < 10 of the enzyme-bound substrate),[18] with the leaving hydroxide group from C3 acting as the base.[19] However, during the syn dehydration of (R)-2-hydroxyacyl-CoA to (E)-2enoyl-CoA catalysed by HAD enzymes (Scheme 1 B), no such mechanism is possible, as the pKa of the hydrogen at C3 is around 40.[4] HADs play a role in the fermentation of various amino acids and (R)-2-hydroxy acids (e.g., d-lactate) in Firmicutes, Fusobacteria and the Archaeon Archaeoglobus fulgidus. Examples of HADs are (R)-lactyl-CoA dehydratase, (R)-2-hydroxyglutaryl-CoA dehydratase, (R)-phenyllactyl-CoA dehydratase and (R)-2-hydroxyisocaproyl-CoA dehydratase.[4, 20] During amino acid fermentation, the growth substrates are first converted into their 2-oxo acids by aminotransferases or dehydrogenases, and are then reduced to the corresponding (R)-2-hydroxy acids. Notably, the latter are always activated to their corresponding CoA esters by CoA-transferases prior to water elimination thus yielding the corresponding (E)-2-enoyl-CoA. The reductive elimination of a hydroxyl group adjacent to a ketone by one-electron donors such as Zn0, CrII or SmII is initiated by the reduction to a ketyl radical. Thereby the electrophilic carbonyl carbon suffers an “umpolung” to a nucleophile, which expels the hydroxyl group. Subsequent one-electron transfer to the transient enoxy radical affords the product.[3] In analogy, is has been postulated that dehydration of 2-hydroxyacyl-CoA involves the one-electron reduction of the thioester carbonyl to a ketyl radical, which eliminates the adjacent hydroxyl group. The resulting enoxy radical, however, is depro 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Architectures of prototypic members of the BCR/HAD family. Similar shades reflects amino acid similarities; similar sizes reflect similar subunits masses. All HgdC-like components are composed of two identical subunits, but the ATP-binding modules of the Azoarcus- (BzdPQ) and Thauera-type (BcrDA) BCRs differ in sequence and subunits mass.
ases;[23] the CoA-ester-binding module is formed of two additional subunits and harbours the active site. In the case of HAD, the two modules do not form a permanent complex,[24] whereas in at least some BCRs a stable complex is formed (Figure 1).[9a] The genes, architectures and cofactors of representative HADs (HgdCAB/LcdCAB/FldIBC/HadIBC)[4] and BCRs (BcrADBC/BadFGDE/BzdQPON)[9b] are listed in Table 1. The two subunits of the electron activation module contain a bridging [4 Fe–4 S] + 1/ + 2 cluster that is coordinated by two cysteines from each subunit.[25] This architecture resembles essentially the Fe-protein of nitrogenases,[26] although there is no marked amino acid sequence similarity. The [4 Fe–4 S] + 1/ + 2 cluster of BCR/HAD is the primary one-electron acceptor from the natural donor, generally a reduced ferredoxin.[27] In HAD the ATP-binding module is a homodimer (~ 28 kDa subunit) and is referred to as “activator” or “initiator” of the dehydratase.[25c] Depending on the type of HAD, the corresponding activator proteins are referred to as HgdC, HadI, LcdC or FldI (Table 1).[4] In BCR, the electron-activating module has a heterodimeric architecture with two similar but different subunits. Among BCRs, two types can be distinguished depending on the level ChemBioChem 2014, 15, 2188 – 2194
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Table 1. Molecular architecture and subunits of prototypic enzymes of the BCR/HAD family of radical enzymes. Organism
Enzyme
Electron-activating module[b] Subunits/GI Mass [kDa] Cofactors
T. aromatica
benzoyl-CoA reductase
BcrA:3724170 BcrD:3724171 BadF:503269128 BadG:503269127 BzdQ:45649069 BzdP:33326777 BzdQ:499555452 BzdP:499555453 HadI:54781340
Rhodopseudomonas palustris benzoyl-CoA reductase
48.4 30.2 47.7 29.2 32.5 28.0 32.4 27.9 28.4
Azoarcus sp. CIB
benzoyl-CoA reductase
Aromatoleum aromaticum
benzoyl-CoA reductase
C. difficile
(R)-2-hydroxyisocaproylCoA dehydratase (R)-2-hydroxyglutarylHgdC:433932 27.3 CoA dehydratase (R)-lactyl-CoA dehydratase LcdC:408407643 27.1
A. fermentans C. propionicum
Clostridium sporogenes Fusobacterium nucleatum
A. fulgidus
28.1
CoA-ester-binding module Subunits/GI Mass [kDa] Cofactors
1 [4 Fe–4 S][25a] BcrB:3724169 BcrC:3724168 1 [4 Fe–4 S][a] BadE:503269129 BadD:503269130 1 [4 Fe–4 S][a] BzdO:45649068 BzdN:45649067 1 [4 Fe–4 S][a] BzdO:499555454 BzdN:499555455 1 [4 Fe–4 S][52] HadB:54781342 HadC:54781343 1 [4 Fe–4 S][44] HgdA:296439332 HdgB:296439333 1 [4 Fe–4 S][a] LcdA:408407641 LcdB:408407642
48.8 43.7 49.5 44.0 50.5 43.4 49.9 43.4 46.3 42.4 53.9 42.0 47.4 41.8
1 [4 Fe–4 S][54]
46.2 43.1 ~ 50 ~ 42 ~ 26
(R)-phenyllactyl-CoA dehydratase (R)-2-hydroxyglutaryl-CoA dehydratase
FldI:16417588
HgdD: 19703551 ~ 27
1 [4 Fe–4 S][a]
(R)-phenyllactyl-CoA dehydratase
HgdC: 1485180
1 [4 Fe–4 S][a]
FldB:16417589 FldC:16417590 HgdA: 19703552 HgdB: 19703553 HgdC: 19703554 HgdA: 1485179 HgdB: 1485178
1 1 1 1 1 1 1 1 2
[4 Fe–4 S][25a] [4 Fe–4 S] [4 Fe–4 S][a] [4 Fe–4 S][a] [4 Fe–4 S][a] [4Fe4S][a] [4 Fe–4 S][a] [4Fe4S][a] [4 Fe–4 S][52]
1 [4 Fe–4 S] FMN, riboflavin[43] 2 [4 Fe–4 S] 0.5 FMN, 0.5 riboflavin[53] 2 [4 Fe–4 S][54] 1 [4 Fe–4 S][55] riboflavin 2 [4 Fe–4 S][a]
[a] Not experimentally verified.
of similarity between the two subunits (Figure 1): 1) Azoarcustype (BzdPQ) is more similar to HAD enzymes with two fairly similar subunits of 28–32 kDa, and 2) Thauera-type (BcrAD or BadFG) with different molecular masses (30 and > 45 kDa).[5, 9b] The CoA-ester-binding modules contain the active site and always comprise two different subunits (42–54 kDa). They generally harbour one or two [4 Fe–4 S] clusters; additionally, some (but not all) HADs contain varying numbers of FMN and riboflavin factors (Table 1, Figure 1).[4] The two active-site subunits show amino acid sequence identities of 26 % (BcrB/HgdA and BcrC/HgdB). Figure 2 shows a phylogenetic tree of the active site-containing BcrC/HgdB subunits of BCR/HAD family members with an amino acid sequence BLAST score higher than 100 (query sequence: BcrC from Thauera aromatica). Cluster I comprises all experimentally verified HgdB-like subunits from 2-hydroxyacyl-CoA dehydratases and many related sequences from Firmicutes, Fusobacteria and Spirochaetes that are proposed to be HAD enzymes. However, there are also HgdB-like sequences from Betaproteobacteria and Actinobacteria; these are not believed to ferment amino acids or 2-hydroxy acids. Cluster II comprises the experimentally verified BCR of the Thauera/Rhodopseudomonas-type (BcrC/BadB from Alpha- and Betaproteobacteria) and Azoarcus-type BCRs (BzdN from Betaproteobacteria, Ferroglobus placidus and a few Deltaproteobacteria). In addition, there are phylogenetic subclusters comprising BcrC-like putative gene products from bacteria that are not known to degrade aromatic compounds under anaerobic conditions, including Clostridia, Bacilli, Actinobacteria, aerobic Alpha- and Betaproteobacteria and methanogenic Archaea. In summary, there are numerous members of the BCR/HAD family with unknown functions, thus indicating that 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
the diversity of this enzyme family is still only poorly understood. A common mechanism of BCRs and HADs? Almost 20 years ago—several years before similarities of the encoding genes of BCRs and HADs were discovered—a common radical mechanism was proposed for both enzyme types.[3] It was based on the one-electron reduction of the thioester functionalities of benzoyl-CoA and the 2-hydroxyacyl-CoA substrates to ketyl intermediates (Scheme 1). In both cases, the redox potentials of this single electron transfer were considered to be far lower than those of any known natural electron donors. For this reason, electron transfer to the thioester has to be coupled the hydrolysis of ATP to ADP and Pi in both BCR and HAD. In the case of benzoyl-CoA, the formed ketyl can be stabilized by several resonance structures over the adjacent aromatic ring (Scheme 1). After protonation to a radical and the second ATP-dependent electron transfer to yield an anion, the second proton transfer yields the final dienoyl-CoA product. In total, two ATP molecules are hydrolysed for the transfer of two electrons.[28] In the case of HAD, umpolung from the nucleophilic thioester to the electrophilic ketyl facilitates the elimination of the adjacent 2-hydroxyl to an enoxy radical (Scheme 1). The pKa of the b-proton of the latter is more than 26 units higher than that of the thioester,[21, 29] and the formed allylic radical can readily be oxidized by the enzyme to the enoyl-CoA product. In summary, the whole process involves one-electron reduction of the thioester substrate by the enzyme and one-electron reChemBioChem 2014, 15, 2188 – 2194
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www.chembiochem.org 500 mV), electron transfer to the aromatic ring would be largely endergonic (DG’ +25 kJ mol 1). For this reason class I BCRs couple the endergonic electron transfer to stoichiometric ATP hydrolysis. All experimental data for the function of BCRs derive from studies with the enzyme from the denitrifying Betaproteobacterium T. aromatica.[9a] The catalytic cycle of this enzyme starts with an electron transfer from reduced ferredoxin (E0’ = 435 mV)[27a] to the [4 Fe–4 S] cluster of the BcrAD subunits 0’ (E 500 mV),[31] thereby reducing it to the +1 state (Scheme 2).[32] In this state, BCR hydrolyses ATP (to ADP + Pi),
Scheme 2. Catalytic cycle of BCR from T. aromatica. ATP/acyl-phosphate hydrolysis switches a [4 Fe–4 S] + 1 cluster from S = 1=2 to the EPR-detectable excited S = 7=2 state (inset). In the presence of substrate, electrons are transferred to the active site; in the absence of substrate, the enzyme falls back to the non-activated state, and the energy dissipates as heat.
Figure 2. Phylogenetic tree of the BCR/HAD family of radical enzymes. The tree was generated from the 283 amino acid sequences with highest similarities to BcrC (T. aromatica). Taxonomic classes are shown in different colours; known subunits of the BCR/HAD family are indicated by arrows. For tree generation, BLAST was used (http://blast.ncbi.nlm.nih.gov/Blast.cgi; expect threshold 10; word size 3; max matches in a query range 0; Matrix: BLOSUM62, Gap costs: existence, 11, extension: 1). Sequences with a BLAST score > 100 were used in Mega5.2 software (http://megasoftware.net/: neighbour joining; 1000 bootstrap replications; Poisson model; pairwise deletion of gaps/missing data). For details see Figure S1 in the Supporting Information.
duction of the enzyme by the transiently formed radical. Hence, the transferred electron is recycled after each turnover, and HAD needs only an initial ATP-dependent one-electron transfer from the activator module. The activated HAD can then catalyse more than 10 000 turnovers before an unspecific reoxidation of the enzyme requires a new round of ATP-dependent activation.[29] In contrast, BCRs depend on stoichiometric ATP hydrolysis for the two one-electron transfer steps to the aromatic substrate.[28]
Functions of BCRs ATP-dependent electron transfer The redox potential of the benzoyl-CoA/cyclohexa-1,5-dienoylCoA redox pair (E0’ = 622 mV) is among the most negative for a substrate/product redox couple in biology.[30] Even with a reduced ferredoxin (E’ under cellular conditions is around 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
with the transient formation of enzyme-derived acyl-phosphate intermediate.[33] Hydrolysis of ATP or acyl-phosphate induces a change of the spin-state of the [4 Fe–4 S] + 1 cluster from the usual S = 1=2 state to the rather unusual S = 7=2 high-spin state, possibly resulting in a marked lowering of its redox potential.[28] In the absence of benzoyl-CoA, the high-energy state switches back to the S = 1=2 ground state, and the energy dissipates as heat.[28] In the presence of benzoyl-CoA, electron transfer occurs from the reduced S = 7=2 cluster to the aromatic substrate via the two [4 Fe–4 S] cluster of the BcrBC subunits. Radical mechanism of substrate reduction Due to a lack of structural information, the mode of benzoylCoA binding in BCR is unknown. Similarities to the corresponding subunits of HAD enzymes (see below) suggest direct ligation to a [4 Fe–4 S] cluster at the carbonyl-oxygen. According to the proposed Birch-like mechanism, two possible transient substrate-based radical species are expected: the radical anion formed by one-electron reduction of benzoyl-CoA and the free radical obtained after protonation of the latter.[16] In single-turnover studies two transient isotropic electron paramagnetic resonance (EPR) signals were detected, with gvalues higher than those of typical organic radicals (g = 2.033 and 2.015), however a clear assignment to substrate-derived radical species was not possible.[34] Kinetic studies with a number of substrate analogues were in accordance with a Birchlike mechanism and suggested that the rate-limiting first electron transfer is probably assisted by a concerted proton transChemBioChem 2014, 15, 2188 – 2194
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CHEMBIOCHEM MINIREVIEWS fer.[35] The most convincing evidence for a Birch-like mechanism comes from two-dimensional NMR studies with labelled substrates.[7] When BCR from T. aromatica reacted with [D5]benzoyl-CoA in H2O, specific anti-dihydro-addition at C3 and C4 occurred. Surprisingly, exchange of a deuteron with the solvent was observed at C2; this can easily be explained with
Scheme 3. Stereochemical course and exchange reaction of BCR from T. aromatica. By using deuterated benzoyl-CoA (1), exchange of the deuteron at C2 is observed; this can be explained by the low pKa of the enoyl-CoA radical (4). Formation of the latter can be explained by protonation of the resonance structures (2 and 3) by a sterically flexible proton donor. Stereospecific anti-dihydro-addition at C3 and C4 leads to either 7 or 8 (which could not be differentiated by NMR).
a radical mechanism shown in Scheme 3. This mechanism allows reversible non-stereospecific protonation at C2, whereby proton addition from one side and proton abstraction from the other explains the observed exchange.[7] In contrast, protonation at C4 is most likely guided by the enzyme with a specific proton donor. BCRs can degrade numerous substrate analogues that are readily reduced by two electrons. In the case of 2-hydroxyand 2-amino-benzoyl-CoA the formed 1,5-dienoyl-products tautomerise/hydrolyse to the 6-oxocyclohex-1-ene-1-carboxylCoA, a common intermediate of the benzoyl-CoA degradation pathway.[9b] In the case of 3-hydroxy-,[36] 3-methyl-[37] and 4methyl-benzoyl-CoA[38] the further catabolism of the dienoylCoA analogues requires a modified benzoyl-CoA degradation pathway. With 3-chlorobenzoyl-CoA as substrate, intrinsic BCR dehalogenase activity was recently identified: the chlorinated substrate is first reduced to the corresponding 3-chloro-1,5-dienoyl-CoA, which then eliminates HCl to yield the rearomatised benzoyl-CoA.[39] A class I BCR-like reaction was also identified in the anaerobic degradation of naphthalene in sulfate-reducing bacteria; here the intermediate 5,6,7,8-tetrahydro-2-naphthoylCoA was reduced to a hexahydronaphthoyl-CoA product in an ATP-dependent manner.[40] 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org Function of HADs ATP-dependent electron transfer Treatment of 2-hydroxyisocaproyl-CoA dehydratase (HadBC) with deazaflavin, EDTA and light (E0’ 650 mV)[41] generated an enzyme with up to 50 % of the activity achieved with the reduced activator HadI in the presence of ATP and Mg2 + (J. Kim and W.B., unpublished results). Hence, for one-electron reduction of the thioester carbonyl to a ketyl radical, a redox potential of about 800 mV could be feasible. As the activator is completely reduced with ferredoxin (E0’ 405 mV),[27b] the redox potential of the activator should be about 300 mV. Thus, hydrolysis of ATP must overcome a difference of ~ 500 mV. By taking DG’ = 50 kJ mol 1 for ATP hydrolysis under physiological conditions (250 mV for the transfer of one electron), two ATP would be sufficient to reach the extremely low potential of 800 mV. The mechanism by which hydrolysis of ATP drives electron transfer from the activator to the dehydratase is still elusive. The four characterised activators share 60–70 % sequence identity and are extremely oxygen-sensitive homodimeric proteins with one [4 Fe–4 S] cluster between the two subunits. The crystal structure of HgdC (3.0 resolution) indeed revealed that the [4 Fe–4 S]2 + cluster is bound at the centre of the activator, coordinated by four cysteines (two from each subunit; Figure 1).[25c] The most remarkable feature of HgdC is that helix 5 of each subunit points towards the cluster, thereby forming a helix-cluster-helix angle of about 1058 (see the similar structure of HadI, Figure 3). The diamagnetic [4 Fe–4 S]2 + cluster is
Figure 3. Views of the activator HadI from C. difficile with bound ADP showing the two subunits (green, blue), [4 Fe–4 S] cluster (yellow (S) and brown (Fe) balls) and ADP (red sticks). Adapted from ref. [13].
easily reduced to [4 Fe–4 S] + with dithionite, TiIII citrate and reduced ferredoxin2 or flavodoxin2 .[27b, 42] EPR spectroscopy revealed spin S = 3=2 for the [4 Fe–4 S] + cluster (g = 4–6) rather than the usual spin (S = 1=2 ), probably due to distortion of the cluster between the two subunits.[43] With excess TiIII citrate, 50 % reduction of HgdC with two electrons to the inactive allferrous state [4 Fe–4 S]0 was achieved.[44] The only other biological example of an all-ferrous cluster is the iron protein of nitrogenase, which has similar properties.[45] ChemBioChem 2014, 15, 2188 – 2194
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CHEMBIOCHEM MINIREVIEWS It has been proposed that upon binding of two ATP molecules to the activator and complex formation with the dehydratase, the angle opens to 1808 and shifts the reduced cluster towards the b-cluster of the dehydratase thus facilitating the one-electron transfer.[4] This hypothesis stems from the crystal structure of the phylogenetically unrelated but functionally similar iron protein of nitrogrenase, which shows almost the same architecture (helix-cluster-helix angle of 1508).[46] In complex with the MoFe protein of nitrogenase stabilized with ADP–AlF3, the angle opened to almost 1808. Although attempts to generate such complexes with HgdC from Acidaminococcus fermentans and HgdAB from Clostridium symbiosum (M. Hetzel and W.B., unpublished results) as well as with HadI and HadBC from Clostridium difficile[24] were successful, diffracting crystals could not be obtained.[25b] Recently crystal structures of reduced HadI in the presence of ADP (1.9 resolution; Figure 3), ADPNP (1.6 ) and ADP-AlF3 (3.0 ) were obtained.[25b] Unexpectedly, the conformational changes induced by the reduced cluster and ligands were very small (and only local), compared to the structure of oxidized HgdC containing ADP. However, taking into account that efficient ATP hydrolysis requires not only the reduced cluster but also the dehydratase,[47] the predicted large conformational change will become visible only in the elusive activator–dehydratase complexes.
Radical mechanism of water elimination The crystal structure of 2-hydroxyisocaproyl-CoA dehydratase (HadBC) from C. difficile immediately gave clues to the mechanism (Figure 4). Each subunit contains one [4 Fe–4 S] cluster coordinated by three conserved cysteines. In the HadB subunit (here referred to as the a-subunit) the ligand of the fourth iron of the a-cluster is a hydroxyl group, whereas in the b-cluster of the b-subunit (subunit HadC) a sulfhydryl group is at this position. Such a ligand has never been detected in other [4 Fe–4 S]
Figure 4. Structure of (R)-2-hydroxyisocaproyl-CoA dehydratase. The a-subunit is coloured according to its three domains. The b-subunit is coloured according to secondary structure: a-helices (blue), b-sheets (orange) and loops (green). The [4 Fe–4 S] clusters with terminal ligands are shown as spheres: sulfur (yellow-green), iron (orange) and oxygen (red). Adapted from ref. [21].
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org clusters. Thus the b-cluster can only act as electron reservoir because there is no possible access for a substrate. In contrast, the a-cluster has access to the protein surface by a channel through which the substrate can approach. Soaking experiments with (R)-2-hydroxyisocaproyl-CoA indeed revealed that the thioester carbonyl replaces the hydroxyl group at the acluster and is hydrogen bonded to a conserved Glu55–Ser37 dyad. The putative mechanism starts with binding of the reduced activator loaded with two ATP molecules at one side of the dehydratase; the closest distance of the activator cluster to the b-cluster is about 12 . Hydrolysis of ATP “shoots” the electron into the b-cluster, which stores the electron until (R)-2-hydroxyisocaproyl-CoA binds to the a-cluster thus replacing the hydroxide at the fourth iron. The removal of several water molecules by the substrate induces an 11 electron transfer from the b- to the a-cluster. Next an inner shell electron transfer to the thioester carbonyl generates the ketyl radical, which (a less efficient Lewis base) leaves the iron and is replaced by the hydroxyl group of the substrate. This “ligand swapping” facilitates the elimination of the hydroxide, which acts as a base to remove the now acidic proton at C3.[21] The stereochemistry of this elimination is consistent with the overall syn geometry of this process.[4] The resulting allylic ketyl radical, which has been identified by EPR spectroscopy,[6] again binds to the fourth iron of the a-cluster and returns its electron, which is used for the next turnover or is stored in the b-cluster.
Outlook Over the past decade, substantial progress in the knowledge of the function of the BCR/HAD family has been achieved. Especially in the case of HAD enzymes, direct spectroscopic evidence of a substrate-based radical and the first crystal structure have largely contributed to a better understanding of the function of this enzyme family. In the case of BCRs, there is still a great need for research to provide evidence of the proposed Birch-like mechanism. For the BCR/HAD family there are perspectives for applied approaches. For example, glutaconic acid production was achieved by introducing six genes from glutamate-fermenting bacteria into Escherichia coli (2-hydroxyglutaryl-CoA dehydratase was the key enzyme).[48] E. coli was converted into a propionate producer by using the genes for lactyl-CoA dehydratase and other enzymes from Clostridium propionicum.[49] With the same promiscuous enzymes adipate can be formed, from which Nylon 6,6 can be produced.[50] Despite its great utility, chemical Birch reduction of aromatic rings has several undesirable attributes, including the handling of alkali metals, cryogenic reaction conditions and the toxicity of the ammonia. For this reasons, there is a high demand for alternatives.[51] The high stereospecificity and the facile conversion of numerous substrate analogues make enzymatic Birch reduction by BCRs an attractive alternative to accomplish stereospecific dihydro additions to aromatic CoA ester substrates. ChemBioChem 2014, 15, 2188 – 2194
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Received: May 28, 2014 Published online on September 9, 2014
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