A catalytically versatile benzoyl-CoA reductase, key enzymein the degradation of methyl- and halobenzoates indenitrifying bacteriaReceived for publication, April 11, 2018, and in revised form, May 15, 2018 Published, Papers in Press, May 16, 2018, DOI 10.1074/jbc.RA118.003329

Oliver Tiedt‡, Jonathan Fuchs‡, Wolfgang Eisenreich§, and X Matthias Boll‡1

From the ‡Fakultät für Biologie–Mikrobiologie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany and §Lehrstuhl fürBiochemie, Technische Universität München, 85747 Garching, Germany

Edited by Ruma Banerjee

Class I benzoyl-CoA (BzCoA) reductases (BCRs) are keyenzymes in the anaerobic degradation of aromatic compounds.They catalyze the ATP-dependent reduction of the centralBzCoA intermediate and analogues of it to conjugated cyclic1,5-dienoyl-CoAs probably by a radical-based, Birch-like reduc-tion mechanism. Discovered in 1995, the enzyme from the de-nitrifying bacterium Thauera aromatica (BCRTar) has so farremained the only isolated and biochemically accessible BCR,mainly because BCRs are extremely labile, and their heterolo-gous production has largely failed so far. Here, we describe aplatform for the heterologous expression of the four structuralgenes encoding a designated 3-methylbenzoyl-CoA reductasefrom the related denitrifying species Thauera chlorobenzoica(MBRTcl) in Escherichia coli. This reductase represents the pro-totype of a distinct subclass of ATP-dependent BCRs that wereproposed to be involved in the degradation of methyl-sub-stituted BzCoA analogues. The recombinant MBRTcl had an����-subunit architecture, contained three low-potential [4Fe-4S] clusters, and was highly oxygen-labile. It catalyzed the ATP-dependent reductive dearomatization of BzCoA with 2.3–2.8ATPs hydrolyzed per two electrons transferred and preferen-tially dearomatized methyl- and chloro-substituted analoguesin meta- and para-positions. NMR analyses revealed that3-methylbenzoyl-CoA is regioselectively reduced to 3-methyl-1,5-dienoyl-CoA. The unprecedented reductive dechlorinationof 4-chloro-BzCoA to BzCoA probably via HCl elimination froma reduced intermediate allowed for the previously unreportedgrowth of T. chlorobenzoica on 4-chlorobenzoate. The heterol-ogous expression platform established in this work enablesthe production, isolation, and characterization of bacterial andarchaeal BCR and BCR-like radical enzymes, for many of whichthe function has remained unknown.

Aromatic compounds are the second most abundant class ofnaturally occurring organic molecules that are predominantlyproduced by plants and microorganisms. Aromatic hydrocar-

bons of fossil oil reservoirs serve as solvents and precursors ofplastics, dyes, resins, insecticides, herbicides, and pharmaceu-ticals, many of which are harmful to the environment andhuman health (1). The complete degradation of aromatic com-pounds by aerobic microorganisms heavily depends on oxyge-nases and has been studied in detail for more than six decades(2, 3). Such a strategy is not an option at anoxic sites, e.g. atmarine or freshwater sediments, aquifers with high carbonloads, or rice fields. In these environments, anaerobic bacteriachannel monocyclic aromatic growth substrates into the cen-tral intermediate benzoyl-CoA (BzCoA).2 It serves as substratefor dearomatizing benzoyl-CoA reductases (BCRs) that dearo-matize their substrate to cyclohexa-1,5-diene-1-carboxyl-CoA(1,5-dienoyl-CoA; see Fig. 1) (4). The redox potential of theBCR substrate/product couple (E�0 � �622 mV) is among thelowest in biology, and electron transfer from any physiologicalreductant to BzCoA, such as reduced ferredoxins (E� � �500mV), has to be coupled to an exergonic reaction (5).

The analogous reaction in organic synthesis, referred to asthe Birch reduction, involves the formation of radical interme-diates at extremely low redox potentials with the first electrontransfer yielding a radical anion being rate-limiting (E�0 � �1.9V for a benzoic acid thioester (6)). The Birch reduction usessolvated electrons as electron donors that are generated by dis-solving alkali metals in ammonia; an alcohol serves as protonsource (7). Kinetic data with BCR favor a Birch-like mechanism(8, 9).

There are two totally nonrelated classes of BCRs, suggestingthey have independently evolved in nature. Class I BCRs pre-dominantly occur in facultative anaerobic bacteria and coupleBzCoA reduction to a stoichiometric ATP hydrolysis (twoATPs hydrolyzed to ADP � Pi per BzCoA reduced). So far, aclass I BCR has exclusively been isolated from the denitrifyingThauera aromatica (BCRTar) more than 20 years ago (10).BCRTar is a 170-kDa heterotetramer with an ����-architec-ture, encoded by the bcrABCD genes (see Fig. 1) (11). Class IIBCRs are found in obligate anaerobes (12, 13). The active site of

This work was supported by the German Research Foundation, DeutscheForschungsgemeinschaft Project BO 1565/14-1. The authors declare thatthey have no conflicts of interest with the contents of this article.

This article contains Figs. S1–S6 and Tables S1–S3.1 To whom correspondence should be addressed: Faculty of Biology,

Institute of Biology II, Schänzlestr. 1, 79104 Freiburg, Germany. Tel.:49-761-2032649; Fax: 49-761-2032626; E-mail: matthias.boll@biologie.uni-freiburg.de.

2 The abbreviations used are: BzCoA, benzoyl-CoA; BCR, BzCoA reductase;MBR, methyl-BzCoA reductase; HAD, 2-hydroxyacyl-CoA dehydratase;UPLC, ultraperformance LC; ESI-Q, electrospray ionization quadrupole; Tar,T. aromatica; Tcl, T. chlorobenzoica; Fpl, F. placidus; Azo, Azoarcus; JTT,Jones–Taylor–Thornton; TES, 2-{[1,3-dihydroxy-2-(hydroxymethyl)-2-pro-panyl]amino}ethanesulfonic acid; TAPS, [tris(hydroxymethyl)methylamino]propanesulfonic acid; HSS, high-strength silica.

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class II BCRs harbors a tungstopterin cofactor (14), and ender-gonic electron transfer to the aromatic ring is suggested to bedriven by a flavin-based electron bifurcation (4).

Class I BCRs belong to the BCR/2-hydroxyacyl-CoA dehy-dratase (HAD) radical enzyme family, which are all composedof two functional modules (16). The dimeric electron-activat-ing module (BcrAD or ��-subunits in BCRTar) harbors an ATP-binding site in each subunit and contains a bridging [4Fe-4S]cluster (6). After reduction of the cluster by a reduced ferre-doxin, ATP hydrolysis results in conformational changes initi-ating low-potential electron transfer to the two [4Fe-4S] clus-ters of the CoA ester– binding module (see Fig. 1). The CoAester– binding module (BcrBC or ��-subunits in BCRTar) cata-lyzes electron transfer to BzCoA. Although class I BCRs trans-fer two electrons and protons to the aromatic ring of BzCoAyielding the reduced 1,5-dienoyl-CoA product (Fig. 1), electrontransfer in the redox-neutral dehydration of 2-hydroxyacyl-CoAs to enoyl-CoAs by HADs is only catalytically required (17,18). The crystal structure of a 2-hydroxyacyl-CoA dehydrataserevealed that the CoA ester substrate is directly ligated to anactive-site cluster via the carbonyl oxygen atom of the thioestersubstrate, suggesting an inner-sphere electron transfer (19). Asimilar mode of binding to an active-site [4Fe-4S] cluster seemsplausible in the catalytic BcrB subunit.

Based on amino acid sequence similarities and subunit sizes,the Thauera and Azoarcus subclasses of class I BCRs are distin-guished. Recently, a phylogenetically distinct third BCR sub-

class has been proposed that comprises BCRs that are up-reg-ulated during complete anaerobic degradation of 4- and3-methylbenzoates (20, 21). Although no BCR of this third sub-class has been isolated and characterized so far, it was suggestedthat MBR-like enzymes are especially optimized for the con-version of para- and meta-substituted BzCoA analogues. Incontrast, BCRTar exhibits a decreased activity with meta-posi-tioned substrates (10, 22, 23), whereas analogues with para-substituents generally do not serve as substrates for BCRTar(22). The only recently reported exception is BCR-catalyzeddefluorination of 4-fluoro-BzCoA to BzCoA (24).

The BCR/HAD family comprises a large number of relatedenzymes of which only a few can unambiguously be assigned totrue BCRs or HADs (16). Heterologous production allowed forthe study of a number of HADs, promoting the elucidation oftheir structure and function (19, 25). Attempts to establish acomparable tool for BCRs were less successful. A first promis-ing step was achieved with the BCR from the hyperthermo-philic euryarchaeon Ferroglobus placidus. Heterologous pro-duction of all four subunits in Escherichia coli yielded arecombinant BCR (BCRFpl) that exhibited BzCoA reducingactivity, albeit 4 orders of magnitude below that of WT BCRTar.This low activity was assigned to the �99% loss of the active-site subunits (26). Recently, functional heterologous expressionof the ATP-binding module of BCRFpl was achieved in theabsence of the active-site subunits (27).

In this work, we developed a general platform for the heter-ologous production of the four subunits of class I BCRs andrelated enzymes. Using this tool, we produced recombinantBCRTar as a proof of principle. Moreover, an archetypical MBRwas heterologously produced and characterized as a class I BCRwith a largely extended substrate preference. This enzymeallowed for the previously unreported growth of Thauera chlo-robenzoica on 4-chlorobenzoate under denitrifying conditions.

Results

Phylogenetic analysis of BCR/HAD family enzymes

The recent discovery of a new subclass of ATP-dependentBCRs putatively specific for the conversion of 3- or 4-methyl-BzCoA (20, 21) motivated us for an updated phylogenetic anal-ysis of the designated active-site subunits of class I BCRs(referred to as BcrB or BzdO). Among the candidates identifiedin databases, only those that derived from gene clusters con-taining all four structural genes of BCRs were considered. Weassessed the diversity by aligning the amino acid sequencesfrom 64 putative BCRs and two HADs (Table S1). In agreementwith earlier studies, the computation of evolutionary distancesrevealed that MBR-like enzymes do not affiliate with Thaueraand Azoarcus subclass BCRs (20, 21). Instead, they group with aseparated cluster of class I BCRs from �,�,�-proteobacteria butalso from a number of distinct phyla (Fig. 2 and Table S1); thisphylogenetic cluster is henceforth referred to as the MBR sub-class of ATP-dependent BCRs.

The analysis identified a putative MBR from T. chloroben-zoica next to the putative 4-methylbenzoyl-CoA reductasefrom Magnetospirillum sp. pMbN1 (20) and the 3-methylben-zoyl-CoA reductase from Azoarcus sp. CIB (21). Similar to

Figure 1. Subunit architecture, cofactors, and electron transfer of BCRTar.The electron-activating module is depicted in gray, and the BzCoA-reducingmodule is depicted in white. The electron transfer from the donor-reducedferredoxin (15) to BzCoA is schematically indicated by arrows. Similarities with2-hydroxyacyl-CoA dehydratases suggest that the cluster in BcrB bindsBzCoA. AH, proton donor for BzCoA reduction.

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Magnetospirillum sp. pMbN1 and Azoarcus sp. CIB, T. chloro-benzoica contains two different class I BCRs, one of the Thau-era subclass (BCRTcl, gi number 1125920966, 1126144411-13)and one of the MBR subclass (MBRTcl, gi number 1126145460-62, 1126145791). The former is highly similar to BCRTar(BcrABCD, 98 –99% amino acid sequence identity), and the lat-ter is highly similar to MBR from Azoarcus sp. CIB (MBRAzo;MbrNOPQ, 96 –98% amino acid sequence identity).

Heterologous production and isolation of BCRTar and MBRTcl

For the development of a heterologous expression platform,the BCRTar-encoding genes bcrABCD and the MBRTcl-encod-ing genes mbrONPQ (also referred to as mbdONPQ in Azoar-cus sp. CIB (21)) were chosen. The corresponding DNAsequences were cloned into the midcopy plasmid pOT1 con-structed in a recent work (28). This plasmid permits induciblegene expression at moderate levels in E. coli to minimize mis-folding of proteins. A C-terminal Strep-Tag II was fused to theindividual �-subunits. After gene expression in E. coli underanaerobic growth conditions, the proteins produced werelargely enriched by affinity chromatography (Fig. 3). The yieldswere 0.4 – 0.6 mg of protein/g of cells.

The four subunits of MBRTcl had an almost perfect 1:1:1:1stoichiometry (Fig. 3A), whereas in preparations of BCRTar the�- and �-subunits of the CoA ester– binding module were fre-quently less represented than the �- and �-subunits of the elec-tron-activating module (Fig. 3B). This finding may result fromstronger contacts between the modules in MBRTcl than inBCRTar under the enrichment conditions.

ATP-dependent BzCoA dearomatizing activities ofrecombinant BCRTar and MBRTcl

Heterologously produced BCRTar and MBRTcl both cata-lyzed the Ti(III) citrate– dependent (5 mM) reduction of BzCoA

to 1,5-dienoyl-CoA as evidenced by coelution with authenticstandards and typical UV/visible spectra. BzCoA reduction ofboth enzymes strictly depended on the presence of MgATP(5 mM); representative diagrams from ultraperformance LC(UPLC)-based assays for MBRTcl are shown in Fig. 4.

BCRTar dearomatized BzCoA to 1,5-dienoyl-CoA at a rate of93 milliunits min�1 mg�1 (1 milliunit � 1 nmol min�1 mg�1).This value is 3– 4 times slower than that reported for the WTenzyme (10), which corroborates the corresponding loss of theactive subunit during the enrichment. The specific activity ofMBRTcl (212 milliunits mg�1) was substantially higher, fittingto the almost stoichiometric presence of all four subunits.Reduced methyl viologen, which routinely served as electrondonor for WT BCRTar in continuous spectrophotometricassays, was also used by recombinant BCRTar at rates compara-ble with that of Ti(III) citrate. However, it barely served as elec-tron donor for MBRTcl (1.5% of the rate with Ti(III) citrate).Addition of 0.5 mM methyl viologen to an assay with 5 mM

Ti(III) citrate as electron donor enhanced BzCoA reductionrate by 10%, indicating that methyl viologen acts as an electroncarrier between Ti(III) citrate and MBRTcl. Sodium dithionite(5 mM) served as an alternative electron donor at 100 (BCRTar)and 53% (MBRTcl) of the rate observed with Ti(III) citrate. Withdithionite as electron donor, UPLC analyses revealed a differentproduct pattern, an observation that was also reported for theconversion of BzCoA by WT BCRTar (29). This altered patternresults from artificial effects of dithionite and its oxidized prod-uct sulfite, yielding physiologically irrelevant CoA ester prod-ucts that were not further analyzed in this work. NAD(P)H didnot serve as electron donor for MBRTcl. Based on the resultsobtained, Ti(III) citrate was routinely used as electron donor inUPLC-based, discontinuous MBRTcl assays.

Native molecular mass, cofactor content, and spectralproperties of MBRTcl

The partial loss of the �- and �-subunits precluded a detailedanalysis of the molecular and kinetic properties of BCRTar, andthey have already been described in detail for WT BCRTar (10).The molecular mass of MBRTcl was 140 � 1 kDa (mean � S.D.

Figure 2. Phylogenetic tree of the BCR/HAD family of radical enzymes.Evolutionary distances of active-site �-subunits calculated by the maximumlikelihood method are illustrated by branch lengths measured in the numberof substitutions per site (scale shown as bar). Phylogenetic assignments cor-respond to the source organisms. Numbers 1– 66 refer to strains listed in TableS1. The asterisk marks MBR from T. chlorobenzoica.

Figure 3. SDS-PAGE of heterologously produced MBRTcl (A) and BCRTar(B) after enrichment by Strep-Tactin affinity chromatography. Lanes arelabeled corresponding to protein fractions obtained during the enrichment:M, protein molecular mass marker; CE, crude extract after cell lysis; MF, mem-brane fraction (150,000 g pellet); SF, soluble fraction (150,000 g superna-tant); FL and EL, flow-through and elution fraction of affinity chromatography,respectively. The subunits of the enriched enzymes are labeled by Greek let-ters. The �-subunit of BCRTar is significantly larger than that of MBRTcl.

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of three biological replicates) as determined by size exclusionchromatography, suggesting a heterotetrameric composition.This value is only slightly lower than that deduced from theamino acid sequence (152 kDa) (25).

The iron content was 11.6 � 1.0 irons per MBRTcl as deter-mined by a spectrophotometric assay, suggesting the presenceof three [4Fe-4S] clusters as described for BCRTar (30). Super-natants of acid-precipitated MBRTcl were analyzed by UPLCcoupled to UV/visible absorbance detection. The resultsobtained excluded the presence of a flavin or other organiccofactors to a significant extent (�0.02 mol/mol of enzyme).

The UV/visible absorbance spectrum of oxidized MBRTclshowed a major maximum at 407 nm and a minor maximum at317 nm (Fig. 5). The spectrum was bleached in the visible regionupon reduction by sodium dithionite at pH 8.3 where E�0 ofdithionite is below �600 mV (31). Titration of MBRTcl withdithionite at this pH revealed an almost linear course of absor-bance decrease at 407 nm up to the addition of one electronequivalent, suggesting that one [4Fe-4S]2�/� cluster was read-ily reduced (Fig. 5C). In this range, the difference spectrum ofoxidized minus dithionite-reduced enzyme revealed a differ-ence absorbance coefficient of �407 � 5.0 mM�1 cm�1 (Fig.5B). Further addition of dithionite did not result in a furtherstoichiometric reduction of the spectrum at 407 nm, indicatingthat a redox equilibrium was approached (Fig. 5C). Upon theaddition of a 300-fold excess of dithionite versus enzyme, themaximal difference in absorbance was reached with �407max �9.6 mM�1 cm�1, indicating the reduction of two of the three[4Fe-4S]2�/� clusters. The difference absorbance at 317 nmcould not be resolved accurately due to the absorbance of accu-mulating dithionite.

Similar to BCRTar, MBRTcl activity was highest at pH �7.3(Fig. S1A). Whereas BCRTar activity was only measurable in arather narrow pH range between 6 and 9 (10), MBRTcl stillexhibited 68% residual activity at pH 6.3 and 88% at pH 8.8.

Sensitivity of MBRTcl toward oxygen

MBRTcl was incubated in air for different time spans beforethe reaction was started with BzCoA under anaerobic condi-tions. The enzyme was severely susceptible to loss of activityupon contact with oxygen with a half-life time in air of around30 s (Fig. S1B). This value is in the same range as half-life timesof WT BCRTar and the activator module of HAD fromAcidaminococcus fermentans, which are 20 and 10 s, respec-tively (10, 32).

Substrate preference of MBRTcl

The substrate preference of MBRTcl was investigated andcompared with reported values for WT BCRTar in UPLC-based

discontinuous assays at 0.25 mM thioester concentrations(Table 1). For selected substrates, the Km, kcat, and kcat/Km val-ues were determined. Most products were additionally sub-jected to electrospray ionization quadrupole (ESI-Q) TOF MSanalysis (Figs. S2–S4).

MBRTcl preferentially dearomatized meta-substituted BzCoAanalogues containing methyl-, chloro-, or hydroxy-functional-ities as indicated by the highest specificity constants deter-mined. With these substrates, relative specific activities com-pared with BzCoA were substantially higher with MBRTcl thanwith BCRTar. Remarkably, MBRTcl also converted para-substi-tuted halo- and methyl-BzCoA analogues that were not con-verted by BCRTar. Exceptions were 3-fluoro- and 4-fluoro-ben-zoyl-CoA that served as substrates for both enzymes. Insummary, MBRTcl appears to be less sensitive to steric effectsof meta- and para-positioned substituents than BCRTar;even 4-bromo-BzCoA was readily converted. Neither of theenzyme converted 4-hydroxy-BzCoA. In contrast, ortho-substituted BzCoA analogues were generally accepted byboth enzymes with BCRTar showing higher relative activitiesthan MBRTcl. Neither of the two enzymes reduced heterocy-clic nicotinoyl-CoA.

ESI-Q-TOF-MS analysis of the products formed from vari-ous ortho-substituted BzCoA analogues by MBRTcl revealed theformation of the corresponding two electron–reduced 1,5-di-enoyl-CoA analogues (Fig. S2). Notably, two different isomerswith the substituent in the 2- or 6-position of the 1,5-dienoyl-CoA products can be formed that cannot be distinguished byMS analysis. The potential 2-hydroxy- and 6-hydroxy-1,5-dien-oyl-CoA isomers formed from 2-hydroxy-BzCoA both sponta-neously tautomerize to 6-oxo-cyclohex-1-enoyl-CoA (6-oxo-1-enoyl-CoA). In the case of the reduction of 3-methyl-,3-fluoro-, and 3-hydroxy-BzCoA analogues, again the corre-sponding 1,5-dienoyl-CoA products were identified with the3-hydroxy-1,5-dienoyl-CoA product most probably again tau-tomerizing to 5-oxo-1-enoyl-CoA. However, 3-chloro- and3-bromo-BzCoA were dehalogenated to BzCoA, which wasthen further reduced to 1,5-dienoyl-CoA (Fig. S3). This findingcan be explained by the spontaneous elimination of HCl/HBrfrom halogenated 1,5-dienoyl-CoA intermediates as reportedearlier for BCRTar (23). Although for 4-methyl-BzCoA the cor-responding 4-methyl-1,5-dienoyl-CoA was identified, 4-fluoro-BzCoA was converted to BzCoA and HF via reductivedefluorination as reported earlier for BCRTar (24). Surprisingly,4-chloro- and 4-bromo-benzoyl-CoA were reductively dehalo-genated by MBRTcl to BzCoA, a reaction that was not observedbefore for any enzyme (Fig. S4).

Figure 4. Conversion of BzCoA to 1,5-dienoyl-CoA by MBRTcl in the absence (A) and presence (B) of MgATP. Shown are UPLC analyses of samples thatwere taken at representative time. 1, BzCoA; 2, 1,5-dienoyl-CoA. AU, absorbance units.

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For further analysis of the product formed from 3-methyl-BzCoA, the preferred substrate of MBRTcl, the enzyme wasincubated with its substrate in D2O, and the product was ana-lyzed by 1H and 13C NMR techniques. The NMR data obtainedare summarized in Table S2; chemical shift maps for the twopossible isomers formed are shown in Figs. S5 and S6. TheNMR data clearly indicate that 3-methyl-1,5-dienoyl-CoA wasspecifically formed by MBRTcl.

ATPase activity of MBRTcl and stoichiometry of ATP hydrolysis

Reduction of BzCoA and analogues of it by MBRTcl strictlydepended on hydrolysis of ATP to ADP � Pi. In the presence ofTi(III) citrate and concurrent absence of a thioester substrate,

the enzyme also exhibited ATPase activity, albeit at only �17%of the rate coupled to substrate reduction (Fig. 6). The stoichi-ometry of ATP hydrolysis and BzCoA reduction was deter-mined by taking samples from a running assay, and the concen-trations of ATP and BzCoA were analyzed by UPLC analysiscoupled to diode array detection in four biological replicates.Using this setup, 2.8 � 0.2 (mean � S.D.) ATPs were hydro-lyzed to ADP � Pi per BzCoA reduced to 1,5-dienoyl-CoA.Assuming that BzCoA-dependent and -independent ATPaseactivities run in parallel, the stoichiometry between ATP hydro-lysis and BzCoA reduction would be 2.3 � 0.2.

Oxygen-sensitive MBRTcl was routinely stored in the pres-ence of 50 �M dithionite. Under anoxic conditions, the enzyme

Figure 5. UV/visible spectra of MBRTcl and titration with dithionite. A, absorption spectrum of oxidized enzyme; the bleaching of the spectra after stepwiseaddition of dithionite is indicated by the arrow at 407 nm. The additionally observed decrease of a shoulder around 320 nm overlapped with the absorbanceof dithionite in the course of the measurement. B, selected difference absorbance spectra of oxidized minus reduced MBRTcl obtained during titration withdithionite. C, titration of MBRTcl with dithionite. Up to the addition of one electron equivalent per protein molecule, an almost linear decrease was observed.Further bleaching required a high excess of dithionite. AU, absorbance units.

Table 1Substrate preference of MBRTcl and BCRTar and identification of the products formedThe relative activities were derived from specific activities determined in assays with 0.25 mM CoA ester substrate and are referenced to those obtained with BzCoA assubstrate (100%). Specific activity of MBRTcl with BzCoA was 212 milliunits mg�1. Activity values of recombinant BCRTar were determined in this work or are referred towildtype BCRTar taken from the references indicated in parentheses after the values. Reaction products were identified by UPLC analysis and, in the case of unknownretention times, and/or UV-visible absorbance spectra coupled to ESI-Q-TOF-MS detection (Figs. S2–S4). After prolonged incubation with Ti(III) citrate, 1,5-dienoyl-CoAand the fluorinated and methylated analogues were further reduced to corresponding monoenoyl-CoAs as reported earlier for BCRTar (29). —, not determined.

SubstrateRelative activities

Kinetic properties ofMBRTcl

Products identifiedMBRTcl BCRTar Km kcat kcat/Km

% �M s�1 s�1 M�1

BzCoA 100 100 10 0.55 5.5 104 1,5-Dienoyl-CoA2-CH3-BzCoA 45 119 — — — 2- or 6-CH3-1,5-Dienoyl-CoA3-CH3-BzCoA 196 97 �5 1.06 2.1 105 3-CH3-1,5-Dienoyl-CoAa

4-CH3-BzCoA 15 �0.1 92 0.10 1.1 103 4-CH3-1,5-Dienoyl-CoA4-CH3-CH2-BzCoA 31 — — — — 4-CH3-CH2-1,5-Dienoyl-CoA2-F-BzCoA 102 145 (22) 66 0.56 8.5 103 2- and 6-F-1,5-Dienoyl-CoA3-F-BzCoA 12 31 (22) — — — 3- and 5-F-1,5-Dienoyl-CoA4-F-BzCoA 7 6 (24) — — — 1,5-Dienoyl-CoA2-Cl-BzCoA 37 10 (22) — — — 2- and/or 6-Cl-1,5-Dienoyl-CoA3-Cl-BzCoA 145 29 �2 0.89 4.4 105 BzCoA4-Cl-BzCoA 18 �0.1 32 0.10 3.1 103 BzCoA2-Br-BzCoA 19 — — — — 2- and/or 6-Br-1,5-Dienoyl-CoA3-Br-BzCoA 63 34 — — — BzCoA4-Br-BzCoA 94 �0.1 — — — BzCoA2-OH-BzCoA 18 — — — — 2- and/or 6-OH-1,5-Dienoyl-CoA3-OH-BzCoA 173 12 (10) — — — 3- and/or 5-OH-1,5-Dienoyl-CoA4-OH-BzCoA �0.1 �0.1 — — — —

a Product was identified by NMR in this study.

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could reversibly be oxidized with a 10-fold excess of thioninewith virtually no loss of activity. In the thionine-oxidized state,CoA ester–independent ATPase activity of MBRTcl was greatlydiminished to �1% of the rate observed in the presence ofBzCoA and Ti(III) citrate. Re-reduction of thionine-oxidizedenzyme by excess Ti(III) citrate fully restored ATPase activityto the level before thionine oxidation. This redox dependenceof the thioester substrate–independent ATPase activity wasalso observed for BCRTar but not for the isolated electron-acti-vating module of the Azoarcus subclass BCR from F. placidus(27, 33, 34).

Complete degradation of 3-methyl- and 4-chlorobenzoate byT. chlorobenzoica

The MBR subclass of ATP-dependent BCRs was originallydeduced from up-regulated bcr-like genes during anaerobicgrowth of Magnetospirillum sp. pMbN1 with 4-methylbenzo-ate (20) and Azoarcus sp. CIB with 3-methylbenzoate (21). Thedearomatizing activities of MBRTcl with 3- or 4-methyl-/chloro-BzCoA analogues motivated us to test whether T. chlo-robenzoica is capable of using the respective benzoate ana-logues as carbon and, together with nitrate, as energy sources.

After three passages, T. chlorobenzoica readily used 3-meth-ylbenzoate and 3-chlorobenzoate as growth substrates underdenitrifying conditions (Fig. 7). Doubling time with the formerwas 3.2 h and only slightly increased versus that observed withbenzoate (2.8 h), whereas that with 3-chlorobenzoate wasclearly higher (6.5 h). No growth with 4-methlybenzoate wasobserved even after incubation for several weeks. In contrast toprevious reports (35, 36), T. chlorobenzoica used 4-chloroben-zoate as a growth substrate under denitrifying conditions with adoubling time of around 18.6 h (Fig. 7). In all cases, an increaseof optical density at 578 nm was observed until the substratewas completely consumed.

Discussion

By establishing the first heterologous production platformfor functional class I BCRs, the recombinant MBR prototype ofan only recently defined BCR subclass was isolated and bio-chemically characterized in this work. The properties are sum-marized and compared with the only other described BCRTarisolated more than two decades ago (Table 2). General proper-ties, such as subunit architecture, cofactor content, ATP depen-

dence/ATPase activity, and oxygen sensitivity, are similar inmembers of different BCR subclasses.

However, there are marked differences between the twoenzymes with regard to substrate specificity. Although BCRTarpredominantly converts BzCoA and ortho-substituted ana-logues, MBRTcl exhibits a clearly extended substrate preferencetoward meta- and para-substituted BzCoA analogues withmethyl, chlorine, and bromine substituents. In particular, thepara-position has previously been suggested as a critical posi-tion for class I BCRs (22), and so far, only the sterically irrele-vant fluorine atom was accepted as a para-substituent byBCRTar, albeit at drastically reduced rates (24).

The observed differential substrate preference of the BCRand MBR subclasses suggests structural variations with regardto the electronic settings of the benzene-binding pocket and thenature, position, and pKa values of amino acid residues involvedin C3 and C4 protonation. The proposed radical anion inter-mediate exhibits highest electron density at the para-position(22). For this reason, any substituent that adds additional elec-tron density at this position should have a counterproductiveeffect on BCR activity as demonstrated by the inability of allBCRs to reduce 4-hydroxy-BzCoA containing a para-substitu-ent having a marked positive mesomeric effect. The positiveinductive effect of alkyl substituents is less pronounced, and asa result MBRTcl dearomatizes 4-methyl-BzCoA, albeit at aclearly reduced rate compared with BzCoA. In contrast, BCRTarshows no activity with 4-methyl-BzCoA, which is most likelydue to steric effects of the relatively bulky methyl moiety. Theproposed steric hindrance of BCRTar by bulky para-substitu-ents is further corroborated by the ability to defluorinate4-fluoro-BzCoAbut not the bulky 4-chloro- and 4-bromo-Bz-CoA analogues with good leaving groups. In contrast, MBRTclconverts para-substituted BzCoA analogues in the order ofbromo � chloro � fluoro as expected from the order of bonddissociation energies (37).

The reductive dehalogenation of 4-chloro-/4-bromo-BzCoAto BzCoA and HCl has not been reported previously for a BCRand accounts for further ATP-dependent dehalogenationreaction of BCRs in addition to the dehalogenation of3-chloro-/3-bromo-BzCoA (23) and 4-fluoro-BzCoA (24). Thedehalogenation reactions most likely proceed via reduced4-chloro-/4-bromo-1,5-dienoyl-CoA intermediates that, simi-

Figure 6. ATPase activities of MBRTcl. ATPase activities were monitored bydetermination of time-dependently formed ADP using UPLC analysis. Shownare ATPase activities of MBRTcl in the thionine-oxidized state and the absenceof BzCoA (E), in the Ti(III) citrate–reduced state and the absence of BzCoA (●),and in the Ti(III) citrate–reduced state and the presence of BzCoA (Œ). Errorbars represent standard deviation.

Figure 7. Anaerobic growth of T. chlorobenzoica with 3-methyl-,3-chloro-, and 4-chlorobenzoate. Growth curves as determined by deter-mination of OD578 of three biological replicates each are depicted as solidlines, substrate consumption curves are depicted as dashed lines. Symbolscorrespond to growth substrates as follows: , 3-methylbenzoate; Œ, 3-chlo-robenzoate; E, 4-chlorobenzoate. Error bars represent standard deviation.

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lar to 3-chloro-1,5-dienoyl-CoA, will spontaneously eliminateHCl/HBr, driven by rearomatization to BzCoA. This assump-tion is supported by the determination of traces of a compoundby ESI-Q-TOF-MS with ion spectra fitting to a chloro-1,5-di-enoyl-CoA intermediate. As an alternative, dehalogenationmay occur on the level of the one electron–reduced aryl radicalanion intermediate. Such an SRN1 mechanism has recentlybeen proposed for aminofutalosine synthase during debromi-nation of a substrate analogue (38).

The preferred substrate of MBRTcl, 3-methyl-BzCoA, waspredominantly reduced to the 3-methyl-1,5-dienoyl-CoA iso-mer, demonstrating the regioselectivity of the enzyme towardthe meta-positioned substituent. This finding is in full accord-ance with the observed complete reductive dehalogenation of3-chloro-BzCoA to BzCoA and HCl in both MBRTcl andBCRTar (23). The latter reaction can only occur via a 3-chloro-1,5-dienoyl-CoA but not via a 5-chloro-1,5-dienyol-CoA inter-mediate. No regioselectivity was observed for the conversion of3-fluoro-BzCoA where both 3- and 5-fluoro-1,5-dienyol-CoAproducts were identified in a recent study (28). Therefore, obvi-ously steric effects govern the regioselectivity of BCRs towardmeta-positioned functionalities.

This work demonstrates that the previously unknown capa-bility of a BCR to reduce 4-chloro-BzCoA to BzCoA and HClallows for growth with 4-chlorobenzoate under denitrifyingconditions. Growth with this halobenzoate in aerobic bacteriahas been demonstrated already 25 years ago (39 –42). Interest-ingly, the aerobic degradation also proceeds via 4-chloro-Bz-CoA, but in this case, dehalogenation is achieved by hydrolysisto 4-hydroxy-BzCoA rather than by reduction, as demon-strated in this work. The 4-hydroxy-BzCoA dehalogenaseinvolved catalyzes a nucleophilic substitution reaction via aMeisenheimer complex, and its structure and function has beenstudied on the molecular level (43–45). Under anaerobic con-ditions, complete mineralization of 4-chloro-benzoate has sofar only been described for enrichment cultures, often compris-ing �-proteobacteria (36, 46 – 49). Thus, T. chlorobenzoicaappears to be the first pure culture described with the capabilityto use 4-chlorobenzoate anaerobically as a growth substrate.

Dechlorination of 4-chloro-BzCoA via ATP-dependentMBR, instead of using a 4-chloro-BzCoA hydrolase, appears atfirst sight as an unnecessary consumption of ATP. However,MBR converts 4-chloro-BzCoA directly to the central interme-

diate BzCoA and circumvents the biosynthesis of 4-hydroxy-BzCoA reductase that would be required for reductive dihy-droxylation. This complex enzyme of the xanthine oxidasefamily depends on numerous redox cofactors, such as molyb-dopterin, FAD, and three Fe–S clusters (11, 50 –52). Finally, theexpense of two ATPs for the reductive dehalogenation appearsto be rather marginal relative to the overall high ATP yieldachieved by the oxidation of benzoate analogues under denitri-fying conditions (53).

The capability of MBRTcl to reduce 4-methyl-BzCoA sug-gests that T. chlorobenzoica is also capable of using 4-methyl-benzoate as a growth substrate as reported for Magnetospiril-lum sp. pMbN1 (20). However, growth of T. chlorobenzoicawith 4-methylbenzoate was not observed, probably due to thelack of a 4-methylbenzoate-CoA ligase and/or downstreamenzymes of the 4-methylbenzoate degradation pathway.

In contrast to BCRTar, MBRTcl did not accept reduced methylviologen as an electron donor, which prevented the use of acontinuous spectrophotometric assay monitoring the oxida-tion of colored reduced methyl viologen. The rationale for thisfinding could be that the redox potential of reduced viologen(E�0 � �446 mV (31)) might be too positive to donate electronsto MBRTcl. Methyl viologen did not act as an inhibitor as thereaction catalyzed by MBRTcl with Ti(III) citrate as electrondonor was stimulated by 0.5 mM methyl viologen.

The stoichiometry of ATP hydrolyzed per electrons trans-ferred is controversial for members of the BCR/HAD family.For 2-hydroxyacyl-CoA dehydratases, a stoichiometry of twoATP hydrolyzed per one electron transferred has been sug-gested (17). In contrast, for BCRTar, stoichiometries of 2.1–2.2ATPs hydrolyzed per BzCoA reduced were determined (33),and for MBRTcl this study revealed a slightly higher stoichiom-etry (2.3–2.8 ATPs hydrolyzed per BzCoA reduced). Takentogether, experimental data point to one rather than two ATPshydrolyzed per electron transferred in class I BCRs, assumingthat each electron is transferred in an ATP hydrolysis– depen-dent manner. The question arises, why the stoichiometriesdetermined for BCRs are always slightly higher than two ATPsper BzCoA? One possible explanation could be that ATP hy-drolysis is not 100% coupled to BzCoA reduction and that aparallel futile background ATP hydrolysis activity exists. Such ascenario is in line with the first electron transfer to the substratebeing rate determining in the overall BCRTar reaction ratherthan ATP hydrolysis (22); it would theoretically allow for a non-coupled ATP hydrolysis to some extent. As an alternativeexplanation, a portion of the active-site module of BCR mightbe inactive, e.g. due to partial cluster degradation. In such ascenario, the apparent ratio of ATP hydrolysis uncoupled ver-sus coupled to BzCoA reduction would be artificially increased.

The expression platform for genes encoding BCRs estab-lished in this work paves the way for the heterologous produc-tion of many other BCR and BCR-like enzymes within the BCR/HAD radical enzyme family. Many BCR-like proteins that donot grow with aromatic compounds under anaerobic condi-tions are present in bacteria (16), and their function hasremained unknown. Very recently, the crystal structure of anATP-dependent, BCR-like enzyme has been solved that con-tained two [4Fe-4S] clusters bridged by an inorganic sulfur

Table 2Comparison of MBRTcl and BCRTar

ND, not determined.Property MBRTcl BCRTar

Reaction catalyzed R–BzCoA3 R–1,5-dienoyl-CoA

R–BzCoA3 R–1,5-dienoyl-CoA

Estimated molecular size (kDa) 140 � 1 168 � 1.3Subunit composition ���� ����Absorption maxima () (nm) 317/407 324/410Reducibility with dithionite

pH 7.3/8.3�/� �/ND

Metal cofactor 11.6 � 1 irons/enzyme 12 irons/enzymeHalf-life time in air (s) 30 20pH optimum 7.3 7.3Relative activity at pH 6/9 (%) �60/�90 �5/�2Reduction of para-substituted

BzCoA analogues4-F-/Cl-/Br-/CH3-

BzCoA4-F-BzCoA

Artificial electron donors, Ti(III) citrate/dithionite/methyl viologen (%)

100/53/1.5 100/100/100

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ligand. The enzyme did not contain a CoA ester– bindingpocket but instead reduced a number of small molecules, suchas acetylene and azide (54). Thus, the catalytic versatility ofBCRs and related enzymes may be much larger than originallyanticipated.

Experimental procedures

Phylogenetic analyses

Amino acid sequences of 66 BcrBTar homologues of the BCR/HAD family (Table S1) were aligned using MUSCLE (55) inMEGA 6.0.6 software. The evolutionary history was inferred byusing the maximum likelihood method based on the JTTmatrix– based model (56). The tree with the highest log likeli-hood (�16,445.6661) was depicted. The initial tree(s) for theheuristic search were obtained automatically by applyingNeighbor-Join and BioNJ algorithms to a matrix of pairwisedistances estimated using a JTT model and then selecting thetopology with superior log likelihood value. The tree was drawnto scale with branch lengths measured in the number of substi-tutions per site. No branch swap filter was applied. All positionscontaining gaps and missing data were eliminated. Evolution-ary analyses were conducted in MEGA 7.0 software.

Growth of bacterial cells

T. chlorobenzoica was cultivated, and the concentrations ofgrowth substrates were determined as described for T. aro-matica in a recent work (24). The initial concentrations ofgrowth substrates and nitrate were 1.3 and 6.0 mM, respectively.

Heterologous gene expression in E. coli

Primers for the amplification of the bcrABCD and mbrONQPgene clusters were derived from partial genome sequences ofT. aromatica and T. chlorobenzoica (gi numbers 19571177 and1341820151, respectively) (Table S3). Reverse primers wereelongated for C-terminal fusions of the �-subunits with Strep-Tag II (SA-WSHPQFEK). Cloning of DNA sequences usingpOT1 expression vector was conducted as described recently(28).

Gene expression was carried out in E. coli MC4100 (57)growing anaerobically in 2 liters of TB medium (89 mM

KH2PO4/K2HPO4 at pH 7.5, 12 g liter�1 Tryptone, 24 g liter�1

yeast extract, 0.4% (v/v) glycerol, 5 mM NaNO3, 50 mM fumar-ate, 0.2 g liter�1 Fe(III) citrate, 75 �g ml�1 kanamycin A). Afterinoculation, flasks were sealed with rubber stoppers, and air inthe headspace was replaced by flushing with N2 gas. Cells wereincubated at 37 °C until reaching an optical density of 0.4 – 0.6whereupon gene expression was induced with 1.0 mM isopropyl�-D-1-thiogalactopyranoside, and growth medium was supple-mented with 1 ml liter�1 vitamin solution VL-7 (58), trace ele-ment solution SL9 (59), and 0.8 mM MgSO4. Cells were incu-bated for another 10 –12 h at 30 °C with agitation at 120 rpm.The pH was monitored and frequently adjusted to values �7.0.Cells were harvested anaerobically by centrifugation at 4,500 g (4 °C) and immediately further processed or stored in liquidnitrogen until use.

Purification of recombinant BCRs

Purification procedures were carried out under anaerobicconditions (95% N2, 5% H2) at 4 °C. Cells were resuspended (1.5ml of buffer/g of cells) in a 20 mM HEPES/NaOH standardbuffer (pH 7.8) containing 200 mM KCl, 4 mM MgCl2, 50 mM

L-arginine, 50 mM L-glutamate, 10% (v/v) glycerol, 1 mM dithio-erythritol, 200 �M dithionite, and �0.1 mg liter�1 DNase I. Cellsuspensions were passed twice through a French pressure cellat 120 megapascals. The cell lysate was centrifuged at150,000 g for 1 h prior to applying the supernatant onto aStrep-Tactin Superflow High Capacity column (IBA) accordingto the manufacturer’s instructions. After washing with DNase-free buffer containing 100 mM KCl and 50 �M dithionite, pro-teins were eluted at pH 7.3 with 3 mM desthiobiotin. Proteinswere concentrated using Vivaspin Turbo 4 centrifugal concen-trators (30,000 molecular weight cutoff) (Sartorius) to finalconcentrations of 5–30 mg ml�1 and stored anaerobically at�80 °C.

Estimation of the molecular mass of MBRTcl

The molecular mass was estimated by size exclusion chroma-tography applying 100 �l of a 3 mg ml�1 protein solution to a24-ml Superdex 200 Increase 10/300 GL column (GE Health-care) in 20 mM HEPES buffer with 150 mM NaCl, 4 mM MgCl2,1 mM dithioerythritol, and 50 �M dithionite. Calibration wasperformed using 100 �l of standard solutions containing thyro-globulin (bovine thyroid) (Mr � 669,000), alcohol dehydroge-nase (Saccharomyces cerevisiae) (Mr � 150,000), carbonicanhydrase (Mr � 29,000), and cytochrome c (equine heart)(Mr � 12,400), each at 3 mg ml�1.

Synthesis of CoA thioesters

BzCoA was synthesized from benzoic acid anhydride andCoA as described (60). Substituted BzCoA analogues were syn-thesized from the corresponding acids via their succinimidylesters as described (61).

Determination of protein and iron content

Protein concentrations were routinely determined by themethod of Bradford (63) using BSA solutions as a standard. Theiron content was determined as described recently (27).Amounts of the heterotetrameric protein applied for determi-nation were 0.5–3 nmol.

UV/visible spectroscopy

UV/visible spectra were recorded under anaerobic condi-tions in a glove box. MBRTcl as isolated was pretreated as amixture of 25.5 �M enzyme plus 125 �M thionine acetate asmild oxidant in 20 mM HEPES buffer containing 4 mM MgCl2and 10% (v/v) glycerol. Thionine was then removed by applyingto a PD MiniTrap G-25 desalting column (GE Healthcare)according to the manufacturer’s instructions. During therecording of UV/visible spectra, 1 ml of MBRTcl diluted to 4.2�M was reduced by addition of dithionite from a 0.5 mM stocksolution in 2.5-�l steps. After the addition of 22.5 �l, the stepswere raised to 10 �l, and after the total addition of 72.5 �l, a 5mM stock solution was used instead.

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Determination of BCR/MBR activities

Enzyme activities were determined, and CoA ester inter-mediates were identified in a discontinuous HPLC/UPLC(Waters)– based assay as described earlier routinely using 5 mM

Ti(III) citrate as artificial electron donor (24, 29). Dithionite,NADH, and dithionite-reduced methyl viologen as alternativeelectron donors were applied at concentrations of 5, 1, and 1mM, respectively. For the determination of the pH dependenceof enzyme activities, MOPS as standard reaction buffer wassubstituted by MES (pH 6.3), TES (pH 7.8), or TAPS (pH 8.3/8.8). Studies of oxygen sensitivity were conducted by aerobicincubation of enzyme solutions for different periods in shaking1.5-ml Eppendorf tubes before the immediate start of reactionsunder anoxic conditions. Reactions were stopped by 2 volumesof methanol, and centrifuged supernatants were subjected toC18 reversed-phase UPLC (Waters). For the detection of lowerCoA ester concentrations during Km value determinations,reactions were stopped by the addition of 0.08 volume of 1%H2SO4, instead. For determination of catalytic constants, theinitial rates determined at different substrate concentrationswere fitted to Michaelis–Menten curves using the Prism 6 soft-ware package (GraphPad). Activity of BCRTar was also deter-mined in a continuous spectrophotometric assay with dithion-ite-reduced methyl viologen serving as the electron donor asdescribed before (10).

Determination of ATPase activities and stoichiometry of ATPhydrolysis/BzCoA reduction

MBRTcl, oxidized as described above for UV/visible spectros-copy, was incubated at a final concentration of 2 �M with 5 mM

ATP and 15 mM MgCl2 in 100 mM MOPS buffer (pH 7.3) at30 °C. Samples were taken at different time points from therunning assays and stopped by the addition of 2 volumes of a 1M HCl solution containing 10% acetonitrile. For determinationof the stoichiometry of ATP hydrolysis and BzCoA reduction,BzCoA was present in the assays. Samples for BzCoA determi-nation were stopped in 66% methanol (v/v, final concentration).

Analysis of ATP/ADP and BzCoA by UPLC was performedusing a Waters C18 HSS T3 column (2.1 100 mm, 1.8-�mparticle size) at 25 °C. ATP/ADP were separated isocratically in10 mM potassium phosphate buffer (pH 6.5) as the sole compo-nent in the mobile phase at a flow rate of 0.2 ml min�1 for 5 min.For detection of BzCoA, the acetonitrile content in 10 mM

potassium phosphate buffer (pH 7) was gradually increased at aflow rate of 0.21 ml min�1 from 2 to 10% within 1.5 min fol-lowed by an increase from 20 up to 30% within 1.6 min. Prod-ucts were identified by comparing retention times and UV/vis-ible absorption spectra with standards and/or additionally bysubsequent MS analysis (see below).

Analysis of CoA esters by MS

LC/MS analysis of CoA esters was performed with a WatersAcquity I class UPLC using a Waters C18 HSS T3 column (2.1 100 mm, 1.8-�m particle size) coupled to a Waters SynaptG2-Si HDMS ESI/Q-TOF system. For separation, a 6-min lin-ear gradient from 2 to 30% acetonitrile in 10 mM ammoniumacetate (pH 6.8) at a flow rate of 0.35 ml min�1 was applied. Themass spectrometer was operated in MS negative or positive

mode with a capillary voltage of 2.5 or 3.0 kV, 120 or 150 °Csource temperature, 400 or 450 °C desolvation temperature,and 750 or 1,000 liters h�1 N2 desolvation gas flow, respectively.In MS negative mode, additionally cone gas flow was applied ata rate of 100 liters h�1.

Analysis of products formed from 3-methylbenzoyl-CoA byNMR spectroscopy

The assay for NMR spectroscopic analyses comprised 0.5 mM

3-methyl-BzCoA, 5 mM Ti(III) citrate, 5 mM MgATP, and 2.5�M MBRTcl in 100 mM MOPS buffer (pH 7.3) with 15 mM

MgCl2 in 95% D2O, and the reaction mixture had a total volumeof 22.7 ml. Reactions were stopped by addition of 66% methanol(v/v, final content), which was removed after subsequent cen-trifugation from the supernatant by flash evaporation at 40 °Cand before freeze-drying the residual solution. CoA thioesterswere purified by reversed-phase HPLC following proceduresdescribed previously (61). Purified compounds were desaltedby solid-phase extraction as described earlier (62).

Prior to analysis, the compounds were dissolved in 0.5 ml ofdeuterated water. 1H NMR and 13C NMR spectra wererecorded at 500 and 126 MHz, respectively, with Avance-HD500 spectrometers operating at 27 °C. 1H-detecting experi-ments, including two-dimensional COSY, NOESY, heteronu-clear single quantum coherence, and heteronuclear multiplebond correlation, were measured with an inverse 1H/13C probehead; direct 13C measurements were performed with a quattronucleus 13C/31P/29Si/19F/1H cryoprobe. All experiments weredone in full automation using standard parameter sets of theTOPSPIN software package (Bruker). 13C NMR spectra wererecorded in proton-decoupled mode. Data processing was typ-ically done with MestreNova software.

Author contributions—O. T. and M. B. conceptualization; O. T.,J. F., W. E., and M. B. formal analysis; O. T. and M. B. supervision;O. T. and M. B. funding acquisition; O. T., J. F., and W. E. validation;O. T., J. F., and W. E. investigation; O. T., J. F., and W. E. visualiza-tion; O. T., J. F., W. E., and M. B. methodology; O. T., J. F., and M. B.writing-original draft; O. T., J. F., and M. B. writing-review and edit-ing; W. E. and M. B. resources; W. E. and M. B. software.

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Oliver Tiedt, Jonathan Fuchs, Wolfgang Eisenreich and Matthias Bollmethyl- and halobenzoates in denitrifying bacteria

A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of

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