Inactive pseudoenzyme subunits in heterotetrameric BbsCD, a novel short‐chain alcohol dehydrogenase involved in anaerobic toluene degradation

Anaerobic toluene degradation proceeds by fumarate addition to produce (R)‐benzylsuccinate as first intermediate, which is further degraded via β‐oxidation by five enzymes encoded in the conserved bbs operon. This study characterizes two enzymes of this pathway, (E)‐benzylidenesuccinyl‐CoA hydratase (BbsH), and (S,R)‐2‐(α‐hydroxybenzyl)succinyl‐CoA dehydrogenase (BbsCD) from Thauera aromatica. BbsH, a member of the enoyl‐CoA hydratase family, converts (E)‐benzylidenesuccinyl‐CoA to 2‐(α‐hydroxybenzyl)succinyl‐CoA and was subsequently used in a coupled enzyme assay with BbsCD, which belongs to the short‐chain dehydrogenases/reductase (SDR) family. The BbsCD crystal structure shows a C2‐symmetric heterotetramer consisting of BbsC2 and BbsD2 dimers. BbsD subunits are catalytically active and capable of binding NAD+ and substrate, whereas BbsC subunits represent built‐in pseudoenzyme moieties lacking all motifs of the SDR family required for substrate binding or catalysis. Molecular modeling studies predict that the active site of BbsD is specific for conversion of the (S,R)‐diastereomer of 2‐(α‐hydroxybenzyl)succinyl‐CoA to (S)‐2‐benzoylsuccinyl‐CoA by hydride transfer to the re‐face of nicotinamide adenine dinucleotide (NAD)+. Furthermore, BbsC subunits are not engaged in substrate binding and merely serve as scaffold for the BbsD dimer. BbsCD represents a novel clade of related enzymes within the SDR family, which adopt a heterotetrameric architecture and catalyze the β‐oxidation of aromatic succinate adducts.


Introduction
Anaerobic toluene degradation is a common property of many unrelated species of denitrifying, metal ion-, or sulfate-reducing bacteria [1][2][3][4][5][6]. In addition, anaerobic phototrophic bacteria and fermentative species in syntrophic cocultures with methanogens are known to metabolize toluene completely as sole carbon source [7,8]. Although these bacteria vary widely with regard to their physiological and phylogenetic affiliation, they use the same principal pathway for anaerobic toluene catabolism.
The initial step of anaerobic toluene catabolism is the highly unusual reaction of benzylsuccinate synthase (BSS; EC 4.1.99.11), which adds the double bond of a fumarate cosubstrate to the methyl group of toluene in a radical-based reaction, producing specifically the (R)-enantiomer of benzylsuccinate ( Fig. 1) [4,6,9,10,11,12,13]. BSS is a glycyl radical enzyme consisting of three subunits and carries two [Fe 4 S 4 ]-clusters per αβγunit [9,14]. The glycyl radical is essential for catalysis and is proposed to initiate the abstraction of a hydrogen atom from toluene via a conserved cysteine in close vicinity at the active site [15][16][17][18][19][20]. BSS is activated to the radical-containing state by the separate activating enzyme BssD (EC 1.97.1.4), which belongs to the family of S-adenosylmethionine-dependent radical enzymes [15] and is encoded in the conserved bss operon together with the genes for the three subunits of BSS and further genes of unknown function ( Fig. 1) [9,21].
β-Oxidation of (E)-benzylidenesuccinate is proposed to continue with an enzyme of the enoyl-CoA hydratase (ECH) family and a short-chain alcohol dehydrogenase, which are encoded in all known bbs operons [12,22]. The bbsH gene codes for an ECH proposed to catalyze the addition of water to the double bond of 2-(E)-benzylidenesuccinyl-CoA to generate the alcohol intermediate 2-(α-hydroxybenzyl)succinyl-CoA (EC 4. 2.1.-). Moreover, the bbs operons always contain two genes coding for apparent subunits of short-chain alcohol dehydrogenases (bbsCD), which are proposed to be involved in the subsequent oxidation of the alcohol intermediate to benzoylsuccinyl-CoA, the keto-intermediate of the pathway (EC 1.1.1.-) [22]. It is unknown whether both gene products or only one constitute the subunits of this enzyme. The last step of benzylsuccinate β-oxidation is catalyzed by a 2-benzoylsuccinyl-CoA thiolase (EC 2.3.1-) encoded by the bbsAB genes [22], which cleaves benzoylsuccinyl-CoA to succinyl-CoA, the CoA-donor for benzylsuccinate activation, and benzoyl-CoA, the central intermediate of anaerobic degradation of most aromatic compounds (Fig. 1).
In addition to toluene metabolism, several other anaerobic degradation pathways have been reported to be initiated by fumarate addition to a methyl group at an aromatic ring in various bacteria, namely, for mxylene [10,20,27], p-cresol [28], 2-methylnaphthalene [29] or p-cymene [30]. In all these cases, bss-and bbslike operons are present in the respective genome The gene products involved are indicated in the same colors as the coding genes above. Note that involvement of BssD (pink) is essential as activating enzyme for BssABC. The intermediates studied in this report are (E)-benzylidenesuccinyl-CoA (1), (S,R)-2-(α-hydroxybenzoyl)-succinyl-CoA (2), and (S)-2-benzoylsuccinyl-CoA (3). Their stereochemistry has been inferred from previous reports [24,25] and this study. Note that the (R)-conformation of the benzylic C-atom in 2 is equivalent to aliphatic (S)-3-hydroxyacyl-CoAs, which is commonly found in β-oxidation pathways. sequences, which code for a BSS-like enzyme and its activating enzyme and for the enzymes of a conserved β-oxidation pathway, respectively, that are involved in degrading the respective compounds [13,31]. In this communication, we report on the biochemical and structural features of the third and fourth enzyme of the β-oxidation pathway for benzylsuccinate, 2-(E)-benzylidenesuccinyl-CoA hydratase (BbsH) and (S,R)-2-(α-hydroxybenzyl)succinyl-CoA dehydrogenase (BbsCD) from T. aromatica.

Synthesis of (E)-benzylidenesuccinate and CoAthioesters
Since (E)-benzylidenesuccinate is not commercially available, it was synthesized chemically from benzaldehyde and dimethylsuccinate as described in methods. The synthetic procedure resulted in 10.6 g of pure (E)benzylidenesuccinic acid with a yield of 78%, which was characterized via its UV/Vis spectrum and behaved identically to the material synthesized previously via a different protocol [24]. The compound was subsequently converted to the internal anhydride, which served as starting compound for the synthesis of thioester derivatives with either CoA or its shortened analogue Nacetylcysteamine (NAC) as described previously [11]. The identity of the produced thioesters was verified via UV-Vis spectroscopy and HPLC analysis. Two product peaks with identical UV-Vis spectra eluting at 2.5 and 3.5 min were detected after conversion with CoA, but only the later eluting compound (43% yield relative to CoA) was turned over by (E)-benzylidenesuccinyl-CoA hydratase and therefore represents the intermediate of the pathway, (E)-2-benzylidenesuccinyl-CoA. The earlier-eluting compound (32% yield relative to CoA) represents the wrong regioisomer, (E)-3benzylidenesuccinyl-CoA. The thioesters were collected separately, lyophilized and stored. While most tests reported in this study were performed with purified (E)-2-benzylidenesuccinyl-CoA, we cannot exclude the presence of traces of the wrong regioisomer or of free CoA in the substrate solution, especially since both thioesters were rather sensitive against hydrolysis and showed increasing decay after storage for more than 3 months. Because of the rapid hydrolysis in neutral or alkaline solutions, enzyme assays with the (E)benzylidenesuccinyl-CoA regioisomers were only possible under mildly acidic conditions (pH < 6.9). Conversion of (E)-benzylidenesuccinic anhydride with NAC yielded a single product peak after HPLC analysis which apparently represents the mixture of both possible regioisomers. As observed for the CoA thioesters, (E)-benzylidenesuccinyl-NAC was rapidly hydrolyzed at pH values > 6.9. Experimental extinction coefficients (ϵ 290 ) of (E)-2-benzylidenesuccinyl-CoA prepared from two batches of independently synthesized benzylidenesuccinic acid were determined as 5600 and 5800 M −1 Ácm −1 , respectively, whereas ϵ 290 of (E)-2-benzylidenesuccinyl-NAC was determined as 7230 M −1 Ácm −1 . Enzyme activities were calculated based on the ϵ 290 values of the preparation used in the respective assay.
Characterization of (E)-2-benzylidenesuccinyl-CoA hydratase (BbsH) All known anaerobic toluene-degrading bacteria contain a gene for a BbsH orthologue in their bbs operons, an ECH implicated in β-oxidation of benzylsuccinate [32,33]. The corresponding gene product is predicted to catalyze the hydration of (E)-2benzylidenesuccinyl-CoA to the corresponding alcohol intermediate, 2-(α-hydroxybenzyl)succinyl-CoA [22]. The bbsH gene from T. aromatica was recombinantly expressed in E. coli BL21(DE3), and the produced protein (UniProt Q9KJE7) was purified via anion exchange chromatography on DEAE-Sepharose and subsequent chromatography on hydroxyapatite. Starting from 5 g of cells (wet mass), 7 mg of purified enzyme were obtained, which showed a specific activity of 27 AE 4 µmolÁmin −1 Á(mg enzyme) −1 , corresponding to a 19-fold enrichment compared to cell extract (for details, see Table 1). In the course of purifying the Table 1. Purification of (E)-2-benzylidenesuccinyl-CoA hydratase (BbsH). Enrichment and yield were calculated based on the apparent enzyme activities in cell extracts of E. coli with overproduced BbsH. Due to thioesterase activities, the actual specific activities are supposed to be higher. Only after the first chromatographic separation, the fractions containing (E)-2-benzylidenesuccinyl-CoA hydratase (called BbsH in the following) activity were free of thioesterase activity as tested by HPLC analysis. The photometric assay to determine BbsH activity was based on the characteristic absorption properties of (E)-2-benzylidenesuccinyl-CoA at 290 nm, which reflect the presence of the double bond in conjugation to the thioester carbonyl group [24]. The double bond is lost during the hydratase reaction, and therefore, the enzyme activity can be monitored by the loss of absorption at 290 nm. Because of the rapid hydrolysis of the thioesters at higher pH values, which leads to an increasing nonenzymatic background of absorption decrease at 290 nm, assays had to be performed in a pH range of 5.8-7.0 and were always accompanied with parallel controls without enzyme. Enzyme activity was based on the rate difference between the assays containing enzyme and the controls. An optimum pH of 6.2 was determined for the reaction, which allowed maximal turnover rates without significant thioester hydrolysis. Product formation from (E)-2-benzylidenesuccinyl-CoA (1) was additionally verified by HPLC analysis, which showed the conversion of the substrate to an earlier-eluting product (2), which had lost absorption at 290 nm ( Fig. 2A,B). Compound 2 showed a main peak at m/z 971.9 in HPLC-MS analysis, which matches the expected mass of 972.7 for the anion of 2-(α-hydroxybenzyl)succinyl-CoA (Fig. 2C).
Enzyme kinetics of BbsH was measured with the natural CoA thioesters as well as with the shorter NAC thioesters of (E)-2-benzylidenesuccinate. The CoA and NAC thioesters were both accepted as substrates and yielded essentially the same results. The obtained data were plotted and analyzed by nonlinear curve fitting, using the equations for Michaelis-Menten and Hill kinetics with or without substrate inhibition. The best fit was obtained with a Hill kinetic model with integrated substrate inhibition according to Eqn 1 [34] (Table 2 and Fig. 2D).
The obtained kinetic parameters showed identical apparent values for both substrates, exhibiting a K 0.5 value of 105 AE 4 µM, a V max of 31.5 AE 0.5 µmol min −1 Ámg −1 ; and a Hill coefficient (N) of 4, accompanied by identical substrate inhibition parameters, represented by a K is constant of 230 AE 4 µM with an exponent (M) of 0.80 AE 0.1. These values indicate that substrate conversion by BbsH is positively cooperative, with a very high Hill coefficient, whereas substrate inhibition shows slightly negative cooperativity. At this point, we cannot exclude that the presence of the wrong regioisomer or free CoA/ NAC in the substrate mixtures may have affected the substrate inhibition or cooperativity parameters.
The native molecular mass of BbsH was determined via size exclusion chromatography and Ferguson plot analysis after native PAGE, yielding apparent native masses of 110 AE 7 kDa and 123 AE 16 kDa, respectively. With a subunit mass of 28 kDa, these data fit best to a BbsH tetramer, but we interpret these data to indicate a trimeric composition of BbsH, because all known members of the well-characterized crotonase/ECH family occur as trimers and hexamers [35][36][37]. Accordingly, a structural model of a BbsH trimer was derived from the trimeric substructure of the methylthioacryloyl-CoA hydratase (DmdD) hexamer with bound substrates, which is involved in dimethylsulfoniopropionate degradation (4IZD; 31% sequence identity) [36] (Fig. 2E). The BbsH model predicts a position of the substrate (E)-benzylidenesuccinyl-CoA in its active site that results in close contacts to the two conserved active site residues E110 and E130 (Fig. 2F).

Characterization of 2-(α-hydroxybenzyl)succinyl-CoA dehydrogenase (BbsCD)
In anaerobic toluene-degrading bacteria, all bbs operons coding for β-oxidation enzymes of benzylsuccinate contain two genes for subunits of a short-chain alcohol dehydrogenase, bbsC and bbsD (UniProt numbers Q9KJF2, Q9KJF1). Accordingly, one or both of the corresponding gene products were postulated to constitute the enzyme oxidizing 2-(α-hydroxybenzyl)succinyl-CoA to 2-benzoylsuccinyl-CoA [22,32]. The bssCD genes from T. aromatica were cloned behind an IPTGinducible trc promotor in pTrc99a and recombinantly expressed in E. coli DH5α, yielding large amounts of BbsC and BbsD in soluble form. The BbsCD complex was then purified via anion exchange chromatography on DEAE-Sepharose and subsequent chromatography on hydroxyapatite, leading to an essentially homogeneous preparation containing both BbsC and BbsD subunits in equal amounts (Table 3). We also constructed an analogous expression vector containing only bbsD with an N-terminal Strep-tag sequence by deletion of the bbsC gene from the expression vector via inverse PCR and religation, but did not obtain any produced protein with this plasmid.
The native molecular mass of BbsCD was determined via gel permeation chromatography and Ferguson plot analysis of native polyacrylamide gels, yielding apparent native masses of 61 AE 5 kDa and 58 AE 3 kDa, respectively. With subunit sizes of 26 kDa (BbsC) and 28 kDa (BbsD), this would best correspond to a dimeric or trimeric structure. However, after determining the actual X-ray structure of the enzyme, the composition of BbsCD was revealed as a stable α 2 β 2 heterotetramer (see below), suggesting that the enzyme showed aberrant migration behavior in the tests used for native size determination.
Neither the substrate nor the product of the proposed reaction is commercially available or can be synthesized via a simple procedure. Therefore, we used a coupled enzyme assay for BbsCD, using the (E)-2benzylidenesuccinyl-CoA hydratase BbsH as auxiliary enzyme to supply the substrate of BbsCD by conversion of (E)-2-benzylidenesuccinyl-CoA. As expected from previous experiments in cell extracts of Thauera aromatica [11], BbsCD accepted nicotinamide adenine dinucleotide (NAD) + , but not NADP + as electron acceptor. Because of the strong sensitivity of the substrate against hydrolysis, the assay had to be performed at slightly acidic pH, and a pH optimum of 6.2 was determined for the coupled assay, the same as for the reaction of BbsH alone. The assays were started with acidic stock solutions of (E)-2-benzylidenesuccinyl-CoA and then monitored for absorbance increase at 365 nm due to NAD + reduction. Remarkably, almost identical BbsCD activities were recorded in the presence or absence of the coupling enzyme BbsH as long as E. coli cell extracts containing BbsCD were used. The reaction Table 2. Kinetic parameters of (E)-2-benzylidenesuccinyl-CoA hydratase with different substrates.
The became only dependent on the addition of BbsH when purified fractions of BbsCD were used, suggesting that an E. coli enzyme unspecifically catalyzed the hydration of (E)-2-benzylidenesuccinyl-CoA. Moreover, the determination of activities in crude extracts was hampered by CoA-thioesterases, leading to less reliable apparent activity values as those measured with purified enzyme batches. Starting from 8.9 g of cells (wet mass), 14 mg of purified BbsCD was obtained, which showed a specific activity of 0.86 AE 0.1 molÁ min −1 Á(mg enzyme) −1 . Due to the competing thioesterases in the cell extract, the calculated enrichment factor of 3.1-fold is certainly underestimated ( Table 3). The final BbsCD fraction was free of contamination by thioesterases or ECHs and homogeneous as judged by SDS/PAGE analysis. The products formed in the coupled assay with BbsH and BbsCD were analyzed by HPLC analysis (Fig. 3A), indicating an initial conversion of (E)-2benzylidenesuccinyl-CoA (1) to 2-(α-hydroxybenzyl) succinyl-CoA (2) catalyzed by BbsH, and the production of another later eluting CoA-thioester (3), which was expected to correspond to 2-benzoylsuccinyl-CoA (Fig. 3A). Our attempts to verify the identity of this new product by HPLC-MS analysis were unsuccessful, but we showed the presence of a keto group in (3) by adding phenylhydrazine (5) to the assay to convert it to a stable hydrazone (4). This resulted in a large shift of the elution position, implying the identity of (3) as benzoylsuccinyl-CoA (Fig. 3A).
Enzyme kinetic measurements with BbsCD were performed using the coupled assay with tenfold excess of BbsH (by activity), and using (E)-2-benzylidenesuccinyl-CoA as starting substrate. We assumed that under these conditions conversion of (E)-2-benzylidenesuccinyl-CoA to 2-(α-hydroxybenzyl)succinyl-CoA is so much faster than the subsequent reaction catalyzed by BbsCD that it does not interfere in the BbsCD kinetics. With this constraint, we observed an apparent saturation kinetics of BbsCD activity with increasing substrate concentration, which was very well described by curve fitting with the Michaelis-Menten equation. The kinetic parameters were determined as apparent K m of 96 AE 8 µM and an apparent V max of 0.96 AE 0.1 µmolÁmin −1 Á(mg protein) −1 (Fig. 3B).
We also screened for potential artificial substrates of BbsCD by assaying the activity of purified enzyme for catalyzing the NADH-dependent reduction of aromatic ketones like acetophenone and chlorinated derivatives, which mimic the natural product benzoylsuccinyl-CoA. BbsCD showed no reducing activity with acetophenone, but two chlorine-containing derivatives, 2,2dichloroacetophene and 2,4 0 -dichloroacetophenone, were reduced with specific activities of 3.5 AE 1 µmolÁmin −1 (mg protein) −1 and 7.0 AE 2 µmolÁmin −1 (mg protein) −1 , respectively. The pH optimum for reduction of these artificial substrates was at pH 6.2, like the one for the forward reaction. However, the kinetic behavior of these reactions only fitted to highly cooperative kinetic models, revealing apparent Hill coefficients of 4 (Fig. 3C).
Structural investigation of BbsCD as a heterotetramer with α 2 β 2 topology The complex structure of BbsCD from T. aromatica with bound NAD + was solved at a resolution of 2.25Å. The final refinement provided a model with R work and R free factors of 0.184 and 0.210, respectively (see Table 4). Electron density is defined for the BbsC chains from G7 to I250 and for the BbsD chains from G2 to G248. Edman sequencing of the two proteins from a proteomic analysis of toluene-induced proteins has revealed before that BbsD lacks the N-terminal methionine, while BbsC had a blocked N terminus [22]. BbsCD is a heterotetramer comprising two BbsC and two BbsD polypeptides. Given that BbsC and BbsD belong both to the SDR protein family, they are structurally very similar with an r.m.s.d. of 0.96Å for 212 C α positions. Despite their common protein fold, the BbsCD tetramer contains only two NAD + molecules, which are bound to the BbsD subunits with well-defined omit electron densities. Unfortunately, we were not able to generate BbsCD crystals, in which the substrate, 2-(α-hydroxybenzyl)succinyl-CoA, or substrate analogues were present. As reported before for  [38], BbsCD is arranged in a symmetry-breaking composition of homodimeric α 2 and β 2 components. Moreover, the subunits BbsC and BbsD are twisted toward each other by 90°in horizontal and 180°in vertical direction (Fig. 4A). All other members of the SDR family with solved structures represent homotetramers [34,39,40,41,42]. The BbsCD subunits comprise a Rossmann fold with its central seven-stranded parallel β-sheet element and six flanking α-helices (Fig. 4B), which is typical for NAD + binding proteins like the members of the SDR family [40,43,44]. Adjacent to the Rossmann fold, four 3 10 -helices are present which follow directly after strands 4-7 of the β-sheet in the sequence. Despite their structural similarity, the BbsC and BbsD subunits differ for their additional α-helices at the periphery of the Rossmann fold: BbsC contains only one (α7), whereas BbsD contains two helices (α6 and α7; Fig. 4B).
Symmetric interactions within the BbsC dimer are mainly stabilized by hydrogen bonds between the α5helices of the respective monomers (S160, G164); in contrast, the BbsD dimer is stabilized by polar interactions of the α4-helices (E103, D107, K115). In both  subunits the first and the second of the 3 10 -helices contribute to structural stabilization by forming interactions with the α5-helix. The asymmetric interactions between the BbsC and BbsD homodimers involve mainly the loop region between α8 and β7 of BbsC1 and the β7 strand with its preceding loop of BbsD1 (and vice versa between BbsC2 and BbsD2).

The BbsCD tetramer harbors two catalytically incompetent BbsC pseudoenzyme subunits
Although the BbsC and BbsD subunits exhibit almost identical Rossmann folds, the BbsC subunits are found by our analyses to act only as built-in pseudoenzyme subunits, as they are unable to bind NAD + or substrate on their own. While the catalytically active BbsD subunits contain the characteristic Ser-Tyr-Lys triad necessary for catalysis in enzymes of the SDR family [40,44,45,46], BbsC is not only missing this conserved sequence motif (see below), but also the GXXXGXG dinucleotide binding motif for NAD + binding (BbsD: G12-G18; [43]) and the conserved aspartate (BbsD: D36) that determines NAD + specificity by interacting with the 2 0 -and 3 0 -hydroxyl group of the adenosine ribose moiety [47] (Fig. 5A). Instead, three bulky residues (D17, E92 and M185) block the active site of BbsC. Calculation of the electrostatic surface potentials of the subunits shows a normal NAD + binding pocket in BbsD, but a highly negatively charged counterpart in BbsC which is incompatible for interacting with the negatively charged diphosphate moiety of NAD + (Fig. 5B).

MD analyses of BbsCDÁbenzoylsuccinyl-CoAÁNADH define stereochemistry of β-oxidation of benzylsuccinate
Given the absence of stereochemical information about the configurations of the BbsCD substrate and product, 2-(α-hydroxybenzyl)succinyl-CoA or benzoylsuccinyl-CoA, we performed molecular dynamics (MD) analysis of the BbsCDÁNADH complex with either (S)-or (R)configured benzoylsuccinyl-CoA to determine its stereoisomer preference. To define initial structures of these ternary BbsCD complexes, we used our structure of the BbsCDÁNAD + complex and the homologous . The latter complex allowed to orient benzoylsuccinyl-CoA in terms of its CoA-moiety as well as the terminal benzoyl group. Using these starting configurations we found that either the (R)-or (S) enantiomers of benzoylsuccinyl-CoA can be principally adopted by the BbsCD binding site. However, when analyzing MD for the different enantiomers of benzoylsuccinyl-CoA (over 3.8 or 5.2 µs, respectively), we observed that (R)benzoylsuccinyl-CoA has a high propensity to dissociate from the BbsCDÁNADH complex (Fig. 6A). In contrast, most trajectories of the BbsCDÁNADHÁ (S)-benzoylsuccinyl-CoA complexes remained stable throughout the whole simulation time (Fig. 6A,B), suggesting that the (S)-enantiomer is the physiological product. Notably, the ligand configuration of (S)-benzoylsuccinyl-CoA is actually the same as of (R)-benzylsuccinyl-CoA (Fig. 1)-the different stereochemical notations are caused by changed priorities of the substituents. Interestingly, the crystal structure of the BbsCDÁNAD + complex shows a glycerol molecule occupying the binding site of the benzoylsuccinyl moiety ( Fig. 6C), which may have interfered with substrate binding in the crystals. Furthermore, the MD-derived BbsCDÁNADHÁ(S)-benzoylsuccinyl-CoA complex shows that the pro-(S) hydrogen of the C4-carbon of NADH points toward the si-side of the carbonyl group of (S)-benzoylsuccinyl-CoA (H Scarbon distance: 2.92 AE 0.17Å), leading to the formation of (S,R)-configured 2-(α-hydroxybenzyl)succinyl-CoA after reductive hydride transfer. From this analysis, we may also infer that this diastereoisomer is the cognate product generated by the previous enzyme of the pathway, BbsH (Figs 1 and 2F) [44,45].

Substrate recognition by BbsCD
The conserved catalytic mechanism within the enzymes of the SDR family involves a catalytic triad generally consisting of S141, Y153 and K157 [35,36,43,47]. These residues are all preserved in BbsD, but not in BbsC (Fig. 6C). The tyrosine plays a major role in the oxidation/reduction reactions, whereas the lysine helps to position the NAD + , and the serine forms hydrogen bonds to the substrate/intermediate/product [44,46,48,49]. Accordingly, in the BbsCDÁNAD + structure K157 forms an intimate hydrogen bond to the 3 0hydroxyl of the nicotinamide nucleotide (3.0Å), and likewise the hydroxyl group of Y153 bonds to the 2 0 -hydroxyl group (2.6Å). Furthermore, our MD simulations show the formation of the expected hydrogen bond between the hydroxyl group of S141 and the carbonyl oxygen of (S)-benzoylsuccinyl-CoA (2.9Å). Other predicted interactions between (S)benzoylsuccinyl-CoA and BbsD include a salt bridge between the product's carboxymethyl moiety and R142 (Fig. 6D, C-C distance between guanidino and carboxyl groups 4.5Å). The phenyl group of (S)benzoylsuccinyl-CoA is positioned in a hydrophobic pocket of BbsD made up by the side chains of M190, W191 and L194. This pocket is created during substrate binding by a blockwise movement of the helical segment S189-E193 toward (S)-benzoylsuccinyl-CoA (Fig. 6B,  D). Interestingly, we did not observe any strong interactions in our MD simulations between the BbsC subunits and the CoA tail of (S)-benzoylsuccinyl-CoA, which might have been expected based on the structures of other 3-hydroxyacyl-CoA dehydrogenases [40,50,51], but we cannot exclude such contacts in the absence of a real structure of the ternary complex.

Phylogenetic analysis of BbsCD
Close orthologues of the BbsCD subunits occur in many known anaerobic hydrocarbon-degrading bacteria, and   branches) and are not affiliated to any of the previously recognized 460 SDR subfamilies [56] (http://sdr-enzy mes.scilifelab.se/SDRfamiliesVer2_20121014.pdf). Most of the BbsC and BbsD sequences even show a similar branching pattern between the respective source organisms, suggesting a long co-evolution of the two adjacent genes (Fig. 7).
BbsH is a typical enzyme of the ECH family, which accepts only one of the (E)-benzylidenesuccinyl-CoA regioisomers and exhibits cooperative enzyme kinetics with substrate inhibition. As known for many enzymes converting CoA thioesters [57], BbsH accepts CoA or NAC thioesters of (E)-benzylidenesuccinate. It is surprising that both substrates are turned over with equal kinetic parameters, but this is consistent with the modeled product binding mode in which the adenosine, phosphate, and pantoic acid residues of CoA remain outside of the binding pocket (Fig. 2E). Like other ECH enzymes, BbsH is predicted to adopt a trimeric quaternary structure with a substrate binding pocket in each subunit (Fig. 2E), whereas a tetrameric composition, as initially suggested by the experimental data, or larger hexameric assemblies as found for DmdD [37], its homology modeling template, can be ruled out. The model of the BbsH:product complex also fits with the predicted reaction mechanism of BbsH (Fig. 1). The two conserved active site residues, E110 and E130, are positioned in H-bonding distance with C2 of the succinyl moiety and the hydroxyl group of the benzylic Fig. 7. Similarity tree of BbsCD and other enzymes of the SDR family. Sequences of the BbsC and BbsD clades (light red/yellow for denitrifying, dark red/green for Fe(III)-or sulfate-reducing hosts) were aligned with SDR enzymes of known functions and structures, some related enzymes from BLAST searches, and representatives of previously annotated SDR subfamilies. Diaminopimelate dehydrogenase (DAP DH) was included as outgroup sequence. All major branches were supported by bootstrapping. Orange branches indicate enzymes oxidizing small substrates such as 1-phenylethanol or 1-(4 0 -hydroxyphenyl)ethanol, hydroxyisoleucine, threonine, 3-or 4-hydroxybutyrate (indicated as Ped, Hped, Hile-DH, HPCoM, Thr-DH, 3-Hbu, and 4-Hbu, respectively), dark blue branches indicate enzymes oxidizing 3-hydroxy-CoA thioesters in degradation pathways of fatty acids or aromatic compounds (BzdZ and Had in benzoate, PaaH in phenylacetate, (S)-Had in fatty acid metabolism, respectively). Green branches indicate enzymes involved in fatty acid or polyhydroxybutyrate biosynthesis (FabG, PhaB, FabL, and FabI, respectively), and the light blue branch indicates enzymes oxidizing sugars of 2-hydroxypropyl-CoM (Hpr-CoM/sugar-DH). Black branches include SDR of unknown function and 150 representatives of the previously described SDR subfamilies [45,56]. Some of the annotated SDR subfamilies containing characterized enzymes are listed. carbon, when (S,R)-2-(α-hydroxybenzyl)succinyl-CoA is modeled into the structure (Fig. 2F). However, despite the apparently good fit, ECH enzymes are often not completely stereospecific and may also exhibit epimerization reactions as side activities [58,59]. Accordingly, predictions of the stereochemical properties of BbsH have to be taken with caution.
BbsCD is an unusual member of the short-chain dehydrogenase/reductase (SDR) family of alcohol dehydrogenases, exhibiting a heterotetrameric α 2 β 2 composition with two active and two inactive subunits. These characteristics are corroborated by the observed necessity of having both genes coexpressed to produce stable recombinant protein BbsCD, because using an otherwise identical expression plasmid containing the bbsD gene without bbsC resulted in complete loss of any protein production. However, only the BbsD subunits contain the conserved amino acids necessary for catalysis as well as for NAD + -binding, whereas all of these residues are missing in BbsC, and some aberrant residues even block access of substrates or cofactors, consistent with the lack of bound NAD + in BbsC in the X-ray structure (Fig. 4A).
In terms of catalysis, BbsCD showed the same relatively low pH optimum as BbsH (pH 6.2), but a normal Michaelis-Menten kinetics, when using (E)-2benzylidenesuccinyl-CoA in the coupled assay with BbsH. The much higher specific activity of BbsH compared with that of BbsCD and the lack of cooperative behavior and substrate inhibition in the coupled assay suggest that BbsCD is not limited by the rate of 2-(αhydroxybenzyl)succinyl-CoA production via BbsH. This implies that the actual activity of BbsCD was recorded without interference of the auxiliary enzyme BbsH. The activities of BbsCD in reducing chlorosubstituted acetophenones indicate its general ability to catalyze the reverse reaction, which could not be assayed with its physiological substrate benzoylsuccinyl-CoA, due to its unavailability. Given that the kinetic data for these substrates cannot be compared to those of the forward reaction, the observed high cooperativity of the reactions may either suggest inhibitory effects of the chlorinated analogues, or potential conformational interactions between the BbsCD subunits.
The structural model of the active site of BbsD with bound NADH and (S)-2-benzoylsuccinyl-CoA is consistent with the general reaction mechanism of alcohol dehydrogenases of the SDR family, which usually catalyze either alcohol oxidation or ketone reduction. The catalytic triad of BbsD differs from that used by other (S)-3-hydroxyacyl-CoA dehydrogenases (Had) (Fig. 7) involved in fatty acid oxidation or other β-oxidation pathways. They employ a conserved triad of Ser, His, and Asn (e. g. S137, H158, N190 in the human enzyme [51]), where the His residue acts as general base [50,51,60]. However, most enzymes of the SDR family contain the same Ser/Tyr/Lys triad as observed in BbsD, for example, the acetoacetyl-CoA reductases (PhaB) producing (R)-3-hydroxybutyryl-CoA for polyhydroxybutyrate (PHB) biosynthesis or secondary alcohol dehydrogenases (Ped, Hped), which appear among the most related SDR clades to BbsD and BbsC (Fig. 7). A scheme of the hydrogen-bonding networks of the residues of the catalytic triad of BbsD with NAD + or NADH and the respective CoA thioesters is shown in Fig. 8. These interactions lead to a local decrease of the pK a value of Y153, allowing it to be deprotonated to a tyrosinate, which acts as base for proton abstraction from (S,R)-2-(α-hydroxybenzyl)succinyl-CoA in a concerted reaction with hydride abstraction by NAD + (Fig. 8). The stereochemistry of substrate and product as determined by MD modeling indicates that the hydrogen of the (R)-configured benzylic C-atom (C α ) of (S,R)-2-(α-hydroxybenzyl)succinyl-CoA is transferred to the reside of the nicotinamide ring of NAD + in the oxidative direction. Conversely, the reaction in reductive direction involves transfer of the pro-(S) hydrogen of NADH to the si-side of the carbonyl group of (S)-benzoylsuccinyl-CoA (Fig. 8). The substrate is intricately bound in a well-defined conformation, involving specific binding sites for the carbonyl-CoA arm, the carboxymethyl substituent, and the phenyl ring (Fig. 6D). Interestingly, the (R)-configuration of the benzylic carbon of (S,R)-2-(α-hydroxybenzyl)succinyl-CoA corresponds to an (S)-configuration of aliphatic 3-hydroxyacyl-CoA substrates (because of different substituent priorities for naming the compounds). (S)-configured 3-hydroxyacyl-CoA intermediates are often found in β-oxidation/ condensation pathways with notable exceptions, for example, for the synthesis of PHB [61]. As revealed by the phylogenetic analysis of SDR enzymes, the stereochemical preference of the enzymes affiliated to different branches does not appear to be highly conserved during evolution. For example, various clades of 1-phenylethanol dehydrogenases shown in Fig. 7 are either specific for the (S)-(Ped/Ped2 or Hped2) or (R)-enantiomers (Hped1 or (R)-Adh) [34,42]. In spite of the large evolutionary distance between BbsCD and standard β-oxidation enzymes, they apparently exhibit common mechanistic properties, such as similar conformation changes of the substrate-binding domain, which tightens around the bound CoA thioesters in an induced-fit mechanism [40].
The unique C2 symmetric arrangement of the BbsCD structure in which dimers of two inactive BbsC and two active BbsD subunits are combined to a heterotetrameric quaternary structure, confirmed an early notion based on sequence data that BbsC subunits do not contribute to catalysis, but merely serve for the structural and/or regulatory integrity of the catalytic BbsD subunits [22]. In the concept of pseudoenzymes, nonfunctional enzyme paralogues regulate catalytic outputs either by protein-protein interactions or by competition for ligands, integrating signaling events, or inducing switching between active and inactive conformations of enzymes or the formation or dissolution of subcellular complexes [62][63][64]. While pseudoenzymes mostly act as separate entities from the enzymes affected, BbsCD is the first example from the SDR family, where the built-in BbsC pseudoenzyme subunits serve as scaffolding and potentially regulatory modules. In this way, they resemble noncatalytic pseudoenzyme-like subunits of some eukaryotic enzymes, for example, 20S proteasomes, plant pyridoxal-5 0 -phosphate synthases, or trypanosomal protein:argininemethyltransferases [62,65,66,67]. The inactive BbsC subunits seem to be important for the functioning of BbsCD, as evident from the observed genetic coupling in operons of every known version of these two genes. The phylogenetic analysis suggests that BbsC and BbsD have originated from a homotetrameric ancestral protein and divergently evolved after functional differentiation, leading to the observed situation of a fully functional BbsD subunit complexed with a BbsC subunit lacking any of the residues involved in catalysis. It is interesting to note that the total absence of conserved residues is only valid for the BbsC subunits from facultative anaerobic denitrifying Betaproteobacteria (labeled yellow in Fig. 7). The highly similar BbsC orthologues from strictly anaerobic Fe(III)-or sulfate-reducing Deltaproteobacteria or Firmicutes (labeled green in Fig. 7) still contain the conserved residues of the catalytic triad and D17, which interacts with the ribose of the adenosyl moiety of NAD + , but have already lost the diphosphate-binding motif. It appears therefore that the BbsC subunits of the latter enzymes may still be in a more preliminary evolutionary state of losing catalytic activity, while those of the enzymes in Betaproteobacteria are fully switched to pseudoenzyme state.

Materials and methods
Synthesis of (E)-benzylidenesuccinyl-thioesters (E)-benzylidenesuccinate was prepared by following a previously described method [68]. Briefly, a solution of 35 mmol benzaldehyde and 44 mmol dimethylsuccinate in 5 mL tert-butanol were added slowly over 4 h under reflux to a mixture of 39 mmol K-tert-butanolate in 25 mL tert-butanol. After additional 3 h of incubation, tert-butanol was evaporated, the precipitate was dissolved in 25 mL 1 M HCl and extracted three times with an equal volume of ethyl acetate. The organic phase was dried by rotating evaporation and dissolved in 15 mL methanol. 25 mL of 15% NaOH in methanol was added and the solution was heated under reflux for 12 h, concentrated by rotating evaporation and resolved in H 2 O (38 mL). After threefold extraction with 25 mL ethyl acetate, the aqueous solution was acidified with HCl to pH 3.0 and again extracted three times with 25 ml ethyl acetate. The organic phases were pooled, dried with Na 2 SO 4 , and concentrated by rotating evaporation. (E)-Benzylidenesuccinate was precipitated from the solution with ethyl acetate and hexane and dried at 60°C. CoA or NAC thioesters of (E)-benzylidenesuccinate were prepared via the internal anhydride as described previously [11,69]. The stability of the thioesters at different pH values or in the presence of extracts containing thioesterase activity was checked in control assays by UV-spectroscopy (loss of absorption at 235 nm) and by HPLC analysis. Hydrolysis of the (E)-benzylidenesuccinyl-thioesters was also visible as loss of UV-absorption at 290 nm in the BbsH enzyme assays.
Cloning of bssH, bbsCD, and bbsD . The bbsCD genes of T. aromatica were amplified via PCR (primers for GGGTCATGAA ATCCAACAGCAATG; rev CGCGGATCCTTCAGCCC GCGAACAGGCTC), cloned into the NcoI/BamHI restriction sites of pTrc99A [70], and expressed from the inducible trc promotor in E. coli DH5α. The resulting plasmid was also modified for expression of only bbsD by deleting the bbsC gene via inverse PCR (Primers CTTTTCGAACTG AGGGTGTGACCATTTCATGGTCTGTTTCCTGTGTG and ATGGGAATTCAGAACAGGGTCGCAC), placing the start codon of bbsD into the same sequence context as that of bbsC in the original plasmid and adding an N-terminal Strep-tag.

Heterologous expression of bssH and bbsCD
The recombinant cells containing the expression plasmids for either bbsH, bbsCD, or bbsD were grown at 20°C in LB medium containing 100 µgÁml −1 ampicillin and expression of the recombinant genes was induced at OD 0.6 with 0.5 mM IPTG. After growth for 14-16 h, cells were harvested by centrifugation and cell extracts were prepared as described elsewhere [71]. BbsH and BbsCD were produced in large amounts, as evident from the appearance of additional protein bands after SDS/PAGE analysis after induction with IPTG, whereas expression of the bssD gene alone did not yield any detectable product, suggesting that the BbsD subunit may not be stable in the absence of BbsC.

Purification of BbsCD
BbsCD was purified chromatographically in two steps. Cell extract (0.75 g total protein) was applied to a DEAE column (30 mL DEAE FastFlow, GE Healthcare, Boston, MA, USA) equilibrated with B1. After washing with 5 CV of B1, the protein was eluted with a linear gradient of 0-150 mM KCl in B1 over 20 CV. BbsCD eluted with 50-100 mM KCl. Active fractions were pooled, diluted with B2 to a salt concentration below 30 mM KCl, and applied to a hydroxyapatite column (20 mL MacroPrep Ceramic Hydroxyapatite Type II, Bio-Rad) equilibrated with B2. After washing the column with 3 CV B2, BbsCD was eluted with a linear gradient of 0-100 mM KCl in B2 over 15 CV. BbsCD eluted at concentrations of 30-40 mM salt.

Protein analytic methods
Native molecular masses of proteins were estimated by gel filtration in relation to the retention volumes of standard proteins (gel filtration calibration kit HMW, GE Healthcare), and by Ferguson plot analysis [72] of the proteins separated by native PAGE (using polyacrylamide concentrations of 6%-10%). Bovine serum albumin (BSA) oligomers and ovalbumin were used as mass standards. Protein concentrations were determined by Coomassie dye binding with BSA as a standard [73]. Separation of proteins by discontinuous SDS/PAGE (13% polyacrylamide) was performed as described previously [74].

Enzyme assays
Catalytic activity of BbsH was determined spectrophotometrically at 30°C by following the decrease in absorption at 290 nm with (E)-benzylidenesuccinyl-CoA or (E)benzylidenesuccinyl-NAC as substrates. The decrease in absorption is caused by the loss of the double bound of the substrate as a result of its hydration with experimentally determined extinction coefficients of 5800 M −1 Ácm −1 and 7230 M −1 Ácm −1 for the CoA and NAC thioester, respectively. The assay mixture (1 mL) contained 50 mM MES/ NaOH pH 6.2 and 0.1-0.3 mg BbsH. The reaction was started with 0.2 mM substrate.

Molecular dynamics analysis of substrate biding by BbsCD
Molecular dynamics simulations of BbsCD using the available crystal structure (PDB: 7PCS) were done with the Amber18 suite [82] using the ff14sb force field for the protein and the TIP3P water model. Parameters for NADH and CoA cofactors were taken from the R.E.DD.B repository (project code F-90) [83]; benzoylsuccinate parameters were derived by antechamber using the Amber GAFF force field. pKa values of ionizable residues of the BbsCD tetramer at pH 6.5 were estimated by H++, but resulted in no deviations from the standard protonation model. For MD simulations, the BbsCD tetramer in complex to 2 NADH and two R-or S-stereoisomers of benzoylsuccinyl-CoA was neutralized by 72 chloride and 94 sodium ions (~0.15 M) and placed in a box of 101.1 x 95.5 x 100.0Å 3 with 27501 water molecules. Initial modeling of the BbsCDÁNADHÁbenzoylsuccinyl-CoA complexes was based on the structure of FabG from Bacillus sp. in complex to acetoacetyl-CoA (PDB code 4NBU). 10 independent BbsCD systems for S-benzoylsuccinyl-CoA (8 for R-benzoylsuccinyl-CoA) were pre-equilibrated over 5.5 ns as NpT ensembles at 300 K with 2-fs steps, 10Å cutoff, and a Monte Carlo barostat. Subsequent trajectories with lengths of 100-600 ns were processed and evaluated in jupyter notebooks using pytraj 2.0.5 (https://github.com/ Amber-MD/pytraj [84]); AmberTools20; and NGLview 2.7.7 [85]). The total length of these production trajectories for the BbsCDÁNADHÁS-benzoylsuccinyl-CoA complex amounts to 5.19 μs, for the BbsCDÁNADHÁR-benzoylsuccinyl-CoA complex to 3.80 μs.

Structural model of BbsH
The structure of BbsH was predicted by homology modeling, using the MODELLER program 10.0 [86] using the structure of methylthioacryloyl-CoA hydratase as template, which exhibits 30% identity to BbsH [37] (PDB 4IZD). Figures were generated using PYMOL 2.1 (DeLano Scientific LLC).