Strains, Plasmids, Growth Conditions, and Molecular Biology Procedures

The E. coli strains and plasmids used in this work are listed in Table 1. The construction of recombinant plasmids featuring the BzdR modules and the chimeras Q1, Q1ΔL and Q2 is detailed in the supplementary materials. E. coli cells were grown on Luria-Bertani (LB) [9] or M63 [10] medium supplemented with the appropriate carbon source at 37°C. Anaerobic growth in LB medium using 10 mM nitrate as the final electron acceptor was achieved as previously reported [7]. Standard molecular biology techniques were performed as described previously [9].

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Table 1. Bacterial strains and plasmids used in this work.

https://doi.org/10.1371/journal.pone.0057518.t001

Enzymatic Assays

β-Galactosidase activities were measured using permeabilized cells as described by Miller [10]. Shikimate kinase assays were performed according to previously established procedures [11] with the modifications detailed below. Plasmid pJCD-P_N was used as as supercoiled P_N template for in vitro transcription assays conducted as previously described [12].

Gel Retardation and DNase I Footprinting Assays

The P_N DNA probe was obtained and mixed with the purified proteins at the concentrations indicated in each assay according to a previously described procedure [7].

Recombinant Plasmid Constructions

To clone the NBzdR, NBzdRL and CBzdR domains, NbzdR, NBzdRL and CbzdR fragments were first PCR amplified from pECOR7 plasmid using the oligonucleotide pairs 5′HisReg (5′-CGGGATCCTTTCCAACGATGAGAACTCATCAC-3′; engineered BamHI site underlined)/N1BzdR (5′-GGGAAGCTTTCACTCCGCCTCCTCGCGCACG-3′; engineered HindIII site underlined), 5′HisReg (see above)/N2BzdR (5′-GGGAAGCTTTCACCTGCGCGCGCTTCGCCCC-3′; engineered HindIII site underlined) and 5′C2 (5′-CGGGATCCTCAGCGTGCCAGGACTTCGAGG-3′; an engineered BamHI is underlined)/3′HisReg (5′-GGGAAGCTTTCAGCGTGCCAGGACTTCGAGG-3′; engineered HindIII site underlined). Next, the PCR amplified products were cloned into the pQE32 vector by BamHI/HindIII double digestion, giving rise to plasmids pQE32-His₆NBzdR, pQE32-His₆NBzdRL and pQE32-His₆CBzdR, respectively. The NbzdR and NBzdRL fragments were also subcloned under the control of the Plac promoter into the pKC01 vector by EcoRI/HindIII double digestionyielding plasmids pCK01NBzdR and pCK01NBzdRL, respectively. The aroK gene that encodes the SKI enzyme was PCR amplified from the E. coli CC118 genome using 5′His-aroK oligonucleotides (5′-CGGGATCCTTGCAGAGAAACGCAATATCTTT-3′; engineered BamHI site underlined)/5′aroKquim (5′-AACTGCAGTTAGTTGCTTTCCAGCATGTGAATA-3′; engineered PstI underlined) and then cloned into the pQE32 vector by BamHI/PstI double digestion, giving rise to plasmid pQE32- His₆SKI.

To construct the Q1 and Q1ΔL chimeras, NBzdR, NBzdRL and aroK were first PCR amplified from pECOR7 plasmid and aroK from the E. coli CC118 genome using the oligonucleotide pairs 5′HisReg (see above)/newN1quim (5′-GGGGTACCCTCCGCCTCCTCGCGCACG-3′; engineered KpnI site underlined) or 5′HisReg (see above)/5′N2qim (5′-GGGGTACCCCTGCGCGCGCTTCG-3`; engineered KpnI site underlined) and 3′aroKqim* (5′-GGGGTACCATGGCAGAGAAACGCAATATCTTT-3′; engineered KpnI site underlined)/5′aroKquim (see above), respectively. The NBzdR and NBzdRL amplified products were cloned into the pGEM-Teasy vector by BamHI/KpnI double digestion, giving rise to plasmids pGEMT-NBzdR and pGEMT-NBzdRL, respectively. The aroK amplified product was then cloned into the pGEMT-NBzdR and pGEMT-NBzdRL vectors by KpnI/PstI double digestion, giving rise to plasmids pGEMT-Q1ΔL and pGEMT-Q1. These constructions were digested using BamHI and PstI enzymes to subclone the Q1ΔL and Q1 genes with N-terminal His₆-tag into the pQE32 plasmid, generating plasmid pQE32-His₆Q1ΔL and pQE32-His₆Q1.

The Q2 chimera containing the Asp168Ala substitution was constructed by directed PCR mutagenesis using pQE32-His₆Q1 plasmid as template. Fragment A spannings from the start codon of aroK to the codon that encodes the Asp36 residue was PCR-amplified with oligonucleotides 3′aroKquim (anneals at the 5′-end of the aroK start codon) and 3′D36A (5′-AATCTCTTGAGCGGAATCGTAAAATTC-3′; anneals at the region spanning codon Asp36 of the aroK gene and replaces it with an Ala codon). Fragment B, spanning from the codon that encodes the Asp36 residue to the 3′-end of aroK, was PCR-amplified with oligonucleotides 5'D36A (5′-GAATTTTACGATTCCGCTCAAGAGATT-3′; anneals at the region spanning codon Asp36 of the aroK gene and replaces it with an Ala codon) and 5'aroKquim (anneals at the 3′-end of aroK). A second round of PCR amplification uses as templates the A and B fragments, and as primers the 3'aroKquim and 5'aroKquim oligonucleotides, which anneal at the start and stop codon of the aroK gene, respectively. The resulting aroK mutant gene that contains the Asp36Ala substitution was KpnI/PstI-double digested, replacing the wild-type aroK gene cloned in pQE32-His₆Q1 to give rise to plasmid pQE32-His₆Q2.

All constructions were confirmed by DNA sequencing.

Shikimate Kinase Activity Assays

Shikimate kinase activity was determined at 25°C by coupling the release of ADP to pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) reactions. Shikimate-dependent oxidation of NADH was monitored at 340 nm (ε = 6180 M⁻¹ cm⁻¹). The standard assay mixture contained (final concentration) 50 mM triethanolamine hydrochloride/KOH buffer, pH 7.0, 50 mM KCl, 5 mM MgCl₂, 1.6 mM shikimate, 2.5 mM ATP, 1 mM phosphoenolpyruvate, 0.1 mM NADH, 3 units of pyruvate kinase (Sigma)/ml, 2.5 units of lactate dehydrogenase (Sigma)/ml, and the target protein to be assayed. K_m values for shikimate were obtained from the Lineweaver-Burk plot [13]. Although it has been described that E. coli SKI has a high K_m (5–20 mM) for shikimate, this value was obtained using an SKI isolated as a heterologous mix of oligomers [14]. Our results show that the His₆-SKI has a K_m for shikimate (0.4 mM) similar to that reported previously for the E. coli SKII [15]. In the competition assays, benzoyl-CoA or AMP were added at a 10 mM concentration to the standard assay conditions, and shikimate kinase activity was compared to that obtained in the absence of these inhibitors.

Fluorescence Assays

Binding of benzoyl-CoA to CBzdR was determined by monitoring the change in protein fluorescence upon ligand addition. Measurements were obtained in an SML-Amicon 8000 spectrophotometer using quartz cuvettes and 0.2-cm excitation and 1-cm emission path lengths (λ_excitation = 275 nm and λ_emission 312 nm; slit widths = 5 nm). Titrations were performed at 25°C by adding a benzoyl-CoA stock solution to 0.4 ml of 20 mM Tris-HCl, pH 8.0, 100 mM KCl containing CBzdR (1 µM initial concentration). The final ligand concentration ranged from 0 to 300 µM. Corrections were made for protein dilutions, background signal, and for the instrument response as indicated by the manufacturer. Control titrations with buffer alone produced no change in the emission signal after correlation. The K_d value for benzoyl-CoA in CBzdR was calculated by non-linear regression using the equation for a one-site saturation model ([L]_b/[P]_t = ([L]_free/K_d+[L]_free) implemented in the GraphPad Prism software (GraphPad software, San Diego); where [L]_b, is the concentration of bound ligand; [P]_t, the total protein concentration, and [L]_free, the concentration of free ligand. The fraction of bound ligand was calculated as (I_o−I)/(I_o−I_min); where I_o is the maximum CBzdR fluorescence intensity (no ligand added); I is the fluorescence intensity after addition of an aliquot of ligand, and I_min is the fluorescence intensity at saturating concentration of ligand (this value was estimated by nonlinear regression to a single hyperbolic decay curve). The data obtained in at least three experimental trials were averaged to calculate the dissociation constant.

NBzdR Is a Functional DNA-Binding Domain

To confirm our hypothesis that the molecular architecture of BzdR arises from the fusion of two individual functional domains, the NBzdR and CBzdR domains were expressed, purified and characterized as independent proteins (Figure 1). Purified NBzdR protein was able to bind to the operator region of the P_N promoter, as illustrated by gel shift (data not shown) and DNase I footprinting experiments (Figure 2A). The expression of NBzdR in E. coli cells with a chromosomal P_N::lacZ fusion elicited the same repression of the P_N promoter observed in cells expressing the entire BzdR regulator (Figure 2B). In vitro transcription experiments also revealed the ability of NBzdR to inhibit the P_N promoter (Figure 2C). The predicted five α helices of NBzdR constitute the minimal structural domain since deletion of helix 5 (residues 77 to 86) did not allow for the production in E. coli of a stable protein that could be detected by SDS-PAGE (not shown).

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Figure 2. The N-terminal domain of BzdR is a functional DNA-binding domain.

A. DNase I footprinting experiments were performed using the P_N probe and the purified regulators His₆-BzdR (control), His₆-NBzdR, and His₆-NBzdRL. The figure shows the results of footprinting assays conducted in the absence of the regulators (lane C), or the presence of 50, 100, or 200 nM of His₆-NBzdR (lanes 1 to 3, respectively) or His₆-NBzdRL (lanes 4 to 6, respectively). Lanes 7 and 8 are footprinting assays containing 50 and 100 nM of purified His₆-BzdR. Lane AG shows the A+G Maxam and Gilbert sequencing reaction. Protected regions (I, II, and III) are indicated with brackets. The −10 box and the transcription initiation site (+1) of the P_N promoter are also shown. Phosphodiester bonds hypersensitive to DNase I cleavage are indicated by asterisks. B. In vivo effect of the N-terminal domain of BzdR on the repression of the P_N promoter. E. coli AFMCPN cells (containing a P_N::lacZ fusion chromosome insertion of the) harboring plasmid pCK01BzdR (BzdR), pCK01NBzdR (NBzdR) or pCK01NBzdRL (NBzdRL) or the control plasmid pCK01 (-), were grown anaerobically in LB medium until the mid-exponential culture phase. β-galactosidase activity is expressed in Miller units. Results from three independent experiments (n = 3) and errors bars are shown. C. Effect of BzdR, NBzdR and NBzdRL on in vitro transcription from P_N. Multiple-round in vitro transcription reactions were performed using the pJCD-P_N plasmid template, which produces a 184-nt mRNA from P_N (arrow), 50 nM E. coli RNAP, and 20 nM Fnr* activator. Transcription reactions were carried out in the absence of repressor (lane -) or presence of 40 nM purified His₆-BzdR (lanes BzdR), His₆-NBzdR (lanes NBzdR) or His₆-NBzdRL (lanes NBzdRL) and in the absence (-) or presence (+) of 2 mM benzoyl-CoA.

https://doi.org/10.1371/journal.pone.0057518.g002

We observed that when NBzdR was fused to the linker region, designated NBzdRL (residues 1 to 130; Figure 1), it was also able to repress the activity of P_N both in vivo and in vitro (Figure 2B and Figure 2C). Moreover, NBzdRL showed greater protection to this promoter (operator regions I and II) than NBzdR and similarly to that observed for complete BzdR protein (Figure 2A). Gel shift experiments also revealed that NBzdRL was able to retard the migration of the P_N promoter at lower concentrations than those required for NBzdR (data not shown). These results suggest that the BzdR linker sequence, whose main function is to facilitate interdomain communication [8], might also play a role in stabilization and binding of the N-terminal domain to the target DNA.

BzdR is a dimer in solution [8]. To test whether this property is conferred by the N-terminal domain, we examined the association state of the NBzdR and NBzdRL proteins. Sedimentation velocity experiments revealed that both proteins sedimented as single species with s-values of 1.8 and 2.3 S, respectively. Also, sedimentation equilibrium experiments confirmed that these species were dimers (Figure S1), demonstrating that BzdR dimerization is an intrinsic property of the N-terminal domain.

We should mention that benzoyl-CoA was unable to rescue the repression caused by NBzdR or NBzdRL over the P_N promoter both in vitro (Figure 2C) and in vivo (data not shown), confirming that NBzdR is not the target of the inducer molecule and, therefore, behaves as a super-repressor. All of these results suggest that NBzdR is a true functional domain that retains the ability of native BzdR protein to bind the P_N promoter and repress its activity.

CBzdR Is a Functional Benzoyl-CoA-Binding Domain

The modelled 3D structure of CBzdR suggests the presence of a deep groove that could interact with the benzoyl-CoA inducer molecule [7], triggering a conformational change in BzdR protein [8]. To establish if effector binding was specific of the C-terminal domain of BzdR, we investigated whether CBzdR would interact with benzoyl-CoA. As expected, fluorescence emission by CBzdR was dramatically modified upon incubation with benzoyl-CoA (Figure S2). Based on the structural model for CBzdR [7], fluorimetry data could be fitted to a single binding site, indicating the formation of a weakly bound CBzdR-benzoyl-CoA complex (K_d 203±35 µM) as previously observed for the parent BzdR regulator (K_d 157±58 µM) [8]. This suggests that benzoyl-CoA induces a conformational change in CBzdR and that it therefore, retains its effector-binding ability in the absence of the NBzdR domain, thus behaving as an independent functional domain.

Analytical ultracentrifugation experiments revealed that CBzdR behaves as a single species with an s-value of 1.8 S, compatible with a globular monomer (Figure S3). The observation that CBzdR is a monomer that undergoes conformational changes in the presence of its cognate ligand (benzoyl-CoA), resembles the behaviour of SKs which undergo extensive conformational changes in the presence of their substrates, i.e., ATP and shikimate [8]. However, despite this conserved similarity with SKs, CBzdR showed no detectable shikimate kinase activity (Figure S4A).

The Q1 Chimera: a Bifunctional Regulator that Controls the P_N Promoter in vitro

The results described above demonstrate that the organization of BzdR is modular and shaped by two functional domains, i.e., NBzdR, which binds to the target promoter and CBzdR, which recognizes benzoyl-CoA as its inducer molecule. This molecular architecture suggests that BzdR may have arisen from the fusion of an HTH-XRE DNA-binding domain to a protein structurally resembling SK or an ancestor of this enzyme. The structural similarity between ATP and shikimate, the two substrates of SK, and the two ends of the benzoyl-CoA molecule, i.e., 3P-ADP and the benzoyl group, suggest that the latter could also be recognized by the SK enzyme. In agreement with this hypothesis, we previously observed that benzoyl-CoA inhibits (by 44%) E. coli SKI activity in a similar manner to AMP (34% inhibition), a well-known SK inhibitor [11], suggesting that benzoyl-CoA can be considered a newly identified ligand of the well-studied SK enzyme.

To unravel the evolutionary pathway that could have given rise to the current BzdR regulator, we used a protein engineering approach and constructed the chimeric protein Q1 by the fusing NBzdRL to E. coli SKI protein (Figure 1). Interestingly, Q1 showed SK activity yielding a K_m for shikimate of 0.6 mM, similar to that exhibited by E. coli SKI purified at our laboratory (K_m 0.4 mM) (Figure S4A). The enzyme activity of the SK domain in the Q1 chimera was also demonstrated in vivo in an E. coli aroK aroL double mutant strain lacking SK activity that is unable to grow in minimal medium since it cannot synthesize aromatic amino acids [11]. E. coli aroK aroL cells transformed with a plasmid that expressed the Q1 protein were observed to grow in glycerol-containing minimal medium (Figure S4B), indicating that Q1 was able to rescue the lack of SK activity in the mutant strain.

In analytical ultracentrifugation experiments, Q1 protein behaved as a single species with an s-value of 3.7 S consistent with a protein dimer (Figure S3). This finding is in agreement with the fact that this chimeric protein contains the NBzdR homodimeric domain, and reveals that dimerization does not prevent the catalytic activity of the monomeric SKI enzyme.

The Q1 chimera was able to bind to the three operator regions of the P_N promoter conferring similar protection to that shown by the BzdR regulator (Figure 3A). In addition, it was capable of inhibiting P_N promoter activity in vitro (Figure 3B, lanes 2-3). Accordingly, we can conclude that both domains are fully and separately active in the Q1 chimera.

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Figure 3. In vitro and in vivo effects of the Q1, Q1ΔL and Q2 chimeras on the P_N promoter.

(A) DNase I footprinting experiments performed out using the P_N probe and the purified regulators His₆-BzdR (control) and His₆-Q1. The figure shows the results of footprinting assays conducted in the absence of the regulators (lane C), or presence of 25, 50, 100, and 200 nM of His₆-BzdR (lanes 1 to 4, respectively) or His₆-Q1 (lanes 5 to 8, respectively). Lane AG shows the A+G Maxam and Gilbert sequencing reaction. Protected regions (I, II, and III) are indicated by brackets. The -10 box and the transcription initiation site (+1) of the P_N promoter are also shown. Phosphodiester bonds hypersensitive to DNase I cleavage are indicated by asterisks. (B) In vitro effect of the Q1 and Q1ΔL chimeras on the activity of P_N. Multiple-round in vitro transcription reactions were performed using the pJCD-P_N plasmid template, which produces a 184-nt mRNA from P_N (arrow), 50 nM E. coli RNA polymerase (RNAP), and 20 nM Fnr* activator [12]. Transcription reactions were conducted in the absence of chimeric regulator (lane 1) or presence of 25 nM (lane 2) or 50 nM (lanes 3–12) of purified His₆-Q1 or His₆-Q1ΔL proteins. Shikimate (S) or shikimate plus ATP (S+ATP) were added at 1 mM (lanes 4 and 7), 2 mM (lanes 5 and 8) or 4 mM (lanes 6, 9, 11 and 12). (C) In vivo effect of BzdR (control) and the Q1 and Q2 chimeras on the activity of the P_N promoter. β-galactosidase activity (in Miller units) of E. coli MC4100 cells harboring plasmid pSJ3P_N (P_N::lacZ) and the plasmids pCK01BzdR (BzdR), pCK01Q1 (Q1), pCK01Q2 (Q2), or the control plasmid pCK01 (-). Cells were grown anaerobically until mid-exponential phase in LB medium supplemented, when indicated, with 5 mM shikimate (S). Results from three independent experiments (n = 3) and errors bars are shown. (D). In vitro effect of the Q2 chimera on the activity of P_N. In vitro transcription reactions were performed as in panel B, in the absence of Q2 (lane 1) or presence of 25 nM (lane 2) or 50 nM (lanes 3–9) of purified His₆-Q2 protein. Shikimate (S) or shikimate plus ATP (S+ATP) were added at 1 mM (lanes 4 and 7), 2 mM (lanes 5 and 8) or 4 mM (lanes 6 and 9).

https://doi.org/10.1371/journal.pone.0057518.g003

It is well known that SK enzymes undergo profound conformation changes when they interact with their substrates, ATP-Mg²⁺ and shikimate. The binding of shikimate leads to a structural state (loaded conformation) that, in the presence of ATP-Mg²⁺, is maintained for a short period of time sufficient to trigger the catalytic reaction to produce 3P-shikimate. The enzyme subsequently recovers its starting structural state (unloaded conformation) (Figure S5) [16], [17]. Since the Q1 chimera is able to bind and inhibit the activity of the P_N promoter in the absence of shikimate and ATP (Figures 3A and 3B), we anticipated that the presence of shikimate alone could provoke a conformational change in the SK domain, i.e., to a loaded-like conformation, that would lead to the release of the Q1 protein from the P_N promoter. When increasing concentrations of shikimate were added to the in vitro transcription assay mix, significant P_N promoter de-repression was observed (Figure 3B, lanes 4–6). Moreover, this de-repression was not observed when the Q1 chimera was replaced with Q1ΔL protein (Figure 3B, lanes 10–12), a new chimera lacking the linker region between the NBzdR and SK domains (Figure 1). These data suggest that the Q1 chimera is a bifunctional regulator that controls P_N promoter activity and responds to shikimate as an inducer molecule in vitro by generating a conformational change transmitted to the DNA-binding domain through the linker region. Interestingly, benzoyl-CoA was also shown to offset the Q1-dependent repression of P_N (Figure 4, lane 5) with an efficiency similar to that shown by shikimate (Figure 4, lane 4) but lower than that observed for the native BzdR (Figure 4, lane 11). As previously noted for BzdR [7], benzoate could not mimic the inducer effect of its CoA derivative (benzoyl-CoA) in the Q1 protein (Figure 4, lane 6), emphasizing the importance of the CoA moiety in effector recognition.

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Figure 4. Effect of different ligands on BzdR-, Q1- or Q2-mediated repression of P_N.

In vitro transcription reactions were run as in Figure 2B, in the absence of repressor proteins (lane 2) or presence of 40 nM purified His₆-Q1 (lanes 3 to 6), His₆-Q2 (lanes 7 to 10) or His₆-BzdR (lanes 11 to 14). The ligands shikimate (S) (lanes 4, 8, 12), benzoyl-CoA (BCoA) (lanes 5, 9, 11), and benzoate (Bz) (lanes 6, 10) were added at 1 mM; ATP was added at 4 mM (lane 13). Lanes -, no ligand added. Lane 1, control assay without RNAP.

https://doi.org/10.1371/journal.pone.0057518.g004

The observation that CBzdR lacks SK activity (Figure S4A) raises the question of whether the existence of such activity in the Q1 chimera may interfere in vivo with effector-dependent de-repression of the P_N promoter. This possibility was confirmed by monitoring P_N promoter activity in E. coli cells expressing the P_N::lacZ reporter fusion and the bifunctional Q1 chimera. It was observed that Q1 protein inhibited the activity of P_N as efficiently as the control BzdR repressor even after the addition of shikimate to the culture medium (Figure 3C). These data strongly suggest that the presence in the cell of both substrates of SK, i.e., shikimate and ATP-Mg²⁺, prevents de-repression of the P_N promoter. The lack of P_N induction by shikimate in vivo was also confirmed in vitro when ATP-Mg²⁺ and shikimate were added together to the transcription assay (Figure 3B, lanes 7–9). These results are in agreement with the rapid conformational changes observed in the SK enzyme after binding of shikimate and ATP-Mg²⁺ during catalysis [18], [19], and suggest the SK domain of the Q1 protein does not remain in the loaded-like conformation (Figure S5) for sufficient time to trigger the release of the repressor from the target promoter.

Taken together, these results confirm that the SK activity of the Q1 regulator prevents effector-specific de-repression of the target promoter by shikimate under physiological conditions.

Evolving the Q1 Chimera towards a BzdR-like Regulator: the Q2 Chimera

The results reported above suggest that CBzdR may have evolved from an SK-like enzyme that lost its enzyme activity while maintaining its benzoyl-CoA binding ability to efficiently control the P_N promoter. To simulate the predicted molecular history of BzdR in the laboratory, we engineered a Q1 derived protein containing a single mutation that abolished its enzyme activity.

Since SK activity is strictly dependent on Mg²⁺, and binding of this metal requires one Thr and two Asp conserved residues [19], we engineered a new synthetic protein, a so-called Q2 chimera (Figure 1), by replacing the Asp168 residue of the Q1 protein (corresponding to Asp36 of SKI) with an Ala residue. This substitution avoids Mg²⁺ binding while maintaining the overall 3D structure of the SK domain. As expected, the purified Q2 protein showed no SK activity (Figure S4A), but was still able to efficiently inhibit the in vitro activity of the P_N promoter (Figure 3D, lanes 2–3). The addition of shikimate or benzoyl-CoA (but not benzoate) to the in vitro transcription reaction released the repression of the P_N promoter (Figure 3D, lanes 4–6; Figure 4, lanes 8–10) suggesting that the SK domain of Q2 retained its ability to bind both ligands and transfer the corresponding conformational changes to the NBzdR domain. Interestingly, shikimate also attenuated the repression exerted by Q2 even in the presence of ATP-Mg²⁺ (Figure 3D, lanes 7–9). Moreover, in vivo experiments revealed that the addition of shikimate to the culture medium of E. coli cells expressing the P_N::lacZ reporter fusion and the Q2 chimera reduced the repression exerted by Q2 (Figure 3C), a behaviour not observed for the Q1 chimera. These results indicate that when the P_N promoter is repressed by the Q2 chimera it can be de-repressed in vivo by shikimate.This observation is consistent with the fact that the catalytically inactive SK domain of the synthetic Q2 regulator can adopt a permanent loaded-like conformation under physiological conditions when shikimate is present, thus triggering the release of the repressor from the target promoter.

All these results point to a role of the Q2 chimera as a synthetic transcriptional regulator that can control the P_N promoter by recognizing shikimate and benzoyl-CoA as inducer molecules. Shikimate and ATP were not effective CBzdR ligands since they were unable to significantly impair the BzdR-dependent inhibition of P_N (Figure 4, lanes 12–13). Thus, the Q2 chimeric protein represents a BzdR-like regulator with a broader effector-binding range and is a likely intermediate in the evolutionary history of the current BzdR protein.

Several lines of research have suggested that early enzyme forms exhibited broad substrate specificity, or “substrate ambiguity”, and later diverged to give rise to the specific enzymes we know today [20], [21], [22], [23]. Proteins acquire new functions without losing their original function, and gene duplication may follow the emergence of a new function, rather than initiate it [24]. In effect, it is known that the SK domain can occur as an independent enzyme, e.g., AroK and AroL proteins in bacteria [15], or as a domain of multifunctional enzymes, e.g., AROM protein in Ascomycetes [25]. Moreover, the duplication of genes coding for SK-like domains is very common in plants and some of these proteins have acquired novel functions, e.g., SKL1, which seems essential for chloroplast biogenesis and SKL2, which encodes a key protein for species-adaptive molecular evolution in Arabidopsis thaliana [26]. Although these duplications are less frequent in prokaryotes, E. coli also has two SKs, and one of these (SKI) could play a role in an as yet unknown cell function since aroK mutants are resistant to mecillinam [27]. In contrast with examples proposed previously whereby an ancestral SK evolved into a novel enzyme, our work shows that the SK domain could also have evolved to give rise to a regulatory protein. We suggest that SK behaves as a “stem” protein domain, able to interact with different substrates and thus facilitating the acquisition of new functions over time. In effect, a similar SK domain has also been detected in a putative regulator (plpp0115 gene product) in the γ-proteobacterium Legionella pneumophila [28].

Concluding Remarks

We report the reconstruction of an analogue of an ancestral protein that could have generated one of the domains of the current BzdR regulator, whose modular architecture comprises two independent functional domains, i.e., a DNA-binding domain (NBzdR) and an effector-binding domain (CBzdR) (Figure 5). The fact that we were able to construct and characterize a bifunctional Q1 regulator strongly suggests that fusion of an HTH-XRE DNA-binding domain to an SK domain was a major step in the evolution of BzdR. In addition, this SK domain was found to behaves as a stem protein domain able to interact with different substrates. The finding that SK recognizes benzoyl-CoA provides additional evidence for our hypothesis. However, the loss of enzyme activity of the SK domain may have been a critical event to improve the efficiency of the ancestral BzdR regulatory protein. Indeed, this was indicated by the catalytically-inactive Q2 chimera which, unlike Q1, was able to respond in vivo to the inducer molecules. This, synthetic Q2 protein constitutes the first shikimate-dependent transcriptional regulator described so far. Our finding that it is possible to construct a transcriptional regulator by fusing an appropriate DNA-binding domain to an enzyme that recognizes the target compound, paves the way for a novel rational approach to create new à la carte regulatory proteins. During the course of evolution of the BzdR regulator, the Q2-like ancestral protein proposed likely suffered additional mutations preventing its recognition of shikimate while increasing its binding affinity for benzoyl-CoA, the inducer and first intermediate of the catabolic pathway controlled by BzdR.

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Figure 5. Proposed model for the evolutionary origin of the BzdR protein.

The red arrows represent fusion between a shikimate kinase ancestor and a DNA-binding domain of the HTH-XRE family. This fusion rendered a protein equivalent to the Q1 chimera. A series of mutations led to the loss of enzyme activity (chimera Q1) and finally to increase in the affinity of the protein for the inducer benzoyl-CoA. Sk: shikimate; 3-P-Sk: 3-phosphoshikimate; Bz-CoA: benzoyl-CoA.

https://doi.org/10.1371/journal.pone.0057518.g005

Besides recreating a plausible pathway for the evolution of an enzyme into a transcriptional regulator, our findings also support the notion that enzymes preceded the emergence of the regulatory systems that control their expression.