The putative l-haloacid dehalogenase gene (DehRhb) from a marine Rhodobacteraceae family was cloned and overexpressed in Escherichia coli. The DehRhb protein was shown to be an l-haloacid dehalogenase with highest activity towards brominated substrates with short carbon chains (≤ C3). The optimal temperature for enzyme activity was 55 °C, and the Vmax and Km were 1.75 μm·min−1·mg−1 of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. DehRhb showed moderate thermal stability, with a melting temperature of 67 °C. The enzyme demonstrated high tolerance to solvents, as shown by thermal shift experiments and solvent incubation assays. The DehRhb protein was crystallized and structures of the native, reaction intermediate and substrate-bound forms were determined. The active site of DehRhb had significant differences from previously studied l-haloacid dehalogenases. The asparagine and arginine residues shown to be essential for catalytic activity in other l-haloacid dehalogenases are not present in DehRhb. The histidine residue which replaces the asparagine residue in DehRhb was coordinated by a conformationally strained glutamate residue that replaces a conserved glycine. The His/Glu dyad is positioned for deprotonation of the catalytic water which attacks the ester bond in the reaction intermediate. The catalytic water in DehRhb is shifted by ~ 1.5 Å from its position in other l-haloacid dehalogenases. A similar His/Glu or Asp dyad is known to activate the catalytic water in haloalkane dehalogenases. The DehRhb enzyme represents a novel member within the l-haloacid dehalogenase family and it has potential to be used as a commercial biocatalyst.
Burkholderia cepacia l-haloacid dehalogenase
Rhodobacteraceae family l-haloacid dehalogenase
Sulfolobus tokodaii l-haloacid dehalogenase
Xanthobacter autotrophicus l-haloacid dehalogenase
- l-DEX YL
Pseudomonas sp. YL l-haloacid dehalogenase
The dehalogenase enzymes catalyse the removal of a halogen atom from an organic substrate and have attracted interest for their applications in bioremediation and in fine chemical production [1, 2]. The production of halogenated by-products from organic synthetic reactions and the use of halogenated pesticides and insecticides in farming are the two main sources of halogenated xenobiotic pollutants. The accumulation of these compounds, which are recalcitrant and carcinogenic pollutants, has driven the search for new microbial dehalogenases suitable for use in bioremediation [2, 3].
Haloacid dehalogenases remove a halogen atom from a carbon adjacent to a carboxyl group. These hydrolytic enzymes can be subdivided into two groups. Group I contains both the nonstereospecific d/l-haloacid dehalogenases and the enantioselective d-haloacid dehalogenases [4, 5]. The enantiospecific l-2-haloacid dehalogenases (l-HADs) that catalyse the dehalogenation of short-chain haloalkanoic acids to produce their corresponding hydroxyalkanoic acids belong to group II. This reaction of group II l-HADs occurs with an inversion of chirality . These enzymes are of particular commercial interest because their products can be used as building blocks for the production of pharmaceuticals and fine chemicals [7, 8].
l-haloacid dehalogenases (EC 188.8.131.52) belong to the HAD enzyme superfamily, which use an aspartate group as the nucleophile in their catalytic mechanism. The HAD superfamily of enzymes also includes phosphate monoesterases, ATPases, phosphonoacetaldehyde hydrolases and phosphomutases . Structurally, these enzymes are dimers with each subunit consisting of two domains. The core domain resembles a ‘Rossmann-fold’ six-stranded parallel β-sheet, which is flanked by five α-helices, and the cap domain consists of a four-helix bundle. The active site is located between these two domains. Proteins in the HAD superfamily share the same core domain but have variable cap domains, which give rise to their enzymatic diversity .
Many l-HADs have been identified, several of which have been biochemically and structurally characterized. These include the l-HADs from Pseudomonas sp. strain YL (l-DEX YL) , Xanthobacter autotrophicus GJ10 (DhlB) , Burkholderia cepacia MBA4 (DehIVa)  and Sulfolobus tokodaii (DehSft) . The structures of the ester intermediates bound to the catalytic aspartate have been determined for l-DEX YL , DhlB  and DehIVa . The structure of a d-lactic acid product bound in the active site of l-DEX YL has also been reported . The structure of l-lactic acid bound in the active site of DehSft  is proposed to mimic the prereaction Michaelis complex. The general SN2 nucleophilic substitution mechanism has been proposed based on the structures of the characterized l-HAD enzymes . The first step of the reaction is the SN2 nucleophilic attack by the catalytic aspartate carboxylate group on the C2 carbon of the substrate, resulting in the formation of an ester intermediate and the release of free halide. The subsequent nucleophilic attack on the ester intermediate by an activated water molecule results in the formation of a d-hydroxyalkanoic acid.
Amino-acid sequence comparison of the l-HADs has highlighted residues that are conserved in all of the biochemically characterized members of the family. Knowledge of the l-HAD crystal structures has allowed assignment of some of these residues to the enzyme active site. Extensive site-directed mutagenesis studies have identified the key amino acids that are essential for catalytic activity. Besides the catalytic aspartate (Asp10) in DehIVa, other completely conserved residues, such as Arg42 and Asn178, are proposed to be involved in substrate binding . The conserved Asp180 in l-DEX YL has been proposed to stabilize the orientation of the catalytic Asp10 and to activate the catalytic water .
To date, the majority of dehalogenase enzymes have been isolated from terrestrial organisms . The marine environment is considered an untapped resource, which is now recognized as an important source of novel enzymes and metabolites . The oceans are known to contain a variety of halogenated compounds, with marine algae being the largest source of organohalogen production [19, 20]. Marine algae produce a diverse range of biologically active compounds, including halogenated terpenes, which are of interest to the pharmaceutical industry because they often show antimicrobial and anticancer properties [21, 22]. Some species of polychaete tube worms are also known to produce a range of structurally diverse halogenated compounds . The toxic characteristics of some halogenated metabolites are thought to be responsible for driving the evolution of algal defence mechanisms [24, 25]. Microorganisms living in symbiosis with tube worms and on the surface of algae may use dehalogenase enzymes to degrade some of these toxic compounds. The abundance of organohalogens in the marine environment makes algae and marine microbes a potential source of novel halogenase and dehalogenase enzymes. A recent study describes a number of species from the Rhodobacteraceae family that tested positive for haloacid dehalogenase activity . However, none of these l-HADs were structurally characterized.
This paper describes the biochemical characterization and structural determination of the Rhodobacteraceae family l-haloacid dehalogenase (DehRhb) enzyme in its native, reaction-intermediate and prereaction Michaelis complex-bound forms. Several amino-acid residues shown to be conserved and essential for activity in related dehalogenases are not present in DehRhb. The amino acids found to be unique to DehRhb are proposed to be involved in the activation of the catalytic water in the second part of the dehalogenase reaction.
Results and Discussion
Cloning, overexpression and purification
A species from the Rhodobacteraceae family that was isolated from a polychaeta worm collected from Tralee beach, Argyll, UK, tested positive for l-HAD activity towards l-monochloropropionic acid (MCPA). Only one gene coding for a putative l-HAD (DehRhb) was identified and had limited sequence identity with known l-HAD enzymes within the 4.5 Mbp genome sequenced as part of this study. The key catalytic aspartic acid residue was conserved, but some other proposed active-site residues in other l-HAD enzymes were not conserved. As DehRhb appeared to be the only candidate gene for the l-HAD, the DehRhb gene was cloned and overexpressed for further characterization. The DehRhb gene was cloned into pET-28a incorporating an N-terminal His-tag. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3)-RIPL, where it accounted for 30% of the total soluble cell protein, as estimated by SDS/PAGE. The recombinant DehRhb enzyme was successfully purified by nickel affinity chromatography (GE Healthcare) and by gel-filtration chromatography using Superdex 75 (GE Healthcare, Buckinghamshire, UK). From the size-exclusion chromatography, the protein was estimated to elute in the form of a dimer. The purified DehRhb enzyme migrated on an SDS polyacrylamide gel at 25 kDa, which agreed with the calculated monomeric protein molecular mass of 25.6 kDa.
Biochemical characterization of the recombinant DehRhb enzyme has shown it to have l-HAD activity. The protein shows varying substrate specificity towards l-2-haloalkanoic acids with different chain lengths and halogen substitutions (Fig. 1). Similarly to previously characterized l-HADs, DehRhb shows activity only towards the l-enantiomer of substrates that are brominated or chlorinated at the α-carbon position.
The DehRhb enzyme shows highest activity towards monobromoacetic acid (100%) followed by monochloroacetic acid (MCAA) (71%), S-bromopropionic acid (71%), S-chloropropionic acid (MCPA) (10%) and dichloroacetic acid (9%). Although activity towards 2-chlorobutryic acid was not observed in the time frame of the substrate-specificity experiment, some activity was observed over 48 h. This shows that DehRhb has reduced activity with substrates that have long carbon chains (>C3).
Both DehRhb and DehIVa (22% amino-acid sequence identity) have a preference for l-brominated substrates with short carbon chains, with highest activity towards monobromoacetic acid and 2-bromopropionic acid . The DhlB enzyme , which has 20% amino-acid sequence identity to DehRhb, also prefers substrates with shorter carbon-chain lengths; however, this enzyme shows highest activity towards dihalogenated substrates that have two halogens attached to the α-carbon, such as dibromoacetic acid and dichloroacetic acid. Other l-HAD enzymes – l-DEX YL  and DehSft  (18% and 19% amino-acid sequence identity to DehRhb, respectively) – show highest activity towards long carbon-chain substrates such as 2-chlorobutryic acid.
The Vmax and Km values for the DehRhb enzyme were calculated to be 1.75 μm·min−1·mg−1 of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. In comparison, l-DEX YL and DehIVa have slightly lower Km values, of 1.1 mm and 1.13 mm, respectively [5, 27]. DehSft has a comparable Km value of 6.23 mm .
The solvent stability of DehRhb was initially assessed by incubating the enzyme in the presence of the solvent at various concentrations for 1 h, as previously carried out for DehSft . In both cases an aliquot of the treated enzyme was then checked for activity in the standard assay. DehRhb showed 150% and 123% activity after incubation in 40% dimethylsulfoxide and 30% methanol, respectively (Fig. 2). This could be explained by the presence of small amounts of these solvents in the final assay conditions that could increase the availability of the substrate (without denaturing the enzyme) as MCAA is more soluble in dimethylsulfoxide and methanol in comparison with water. The DehRhb enzyme retained 100% of its activity after incubation in 20% ethanol and 10% acetonitrile.
Solvent-stability experiments with DehRhb were also carried out by performing thermal shift experiments with the enzyme in the presence of different solvents over a range of concentrations. The results of these experiments (Table 1) showed that in the presence of 10–40% ethanol, methanol and acetonitrile the enzyme melting temperature decreased by between 6 and 34 °C. No melting temperature was recorded for 50% ethanol, methanol and acetonitrile, suggesting that the enzyme had unfolded at these concentrations of solvent. The enzyme was most stable in 10–50% dimethylsulfoxide, showing a decrease in melting temperature of between 6 and 22 °C.
|Concentration of solvent (%)||Melting temperature in ethanol (°C)||Melting temperature in methanol (°C)||Melting temperature in acetonitrile (°C)||Melting temperature in dimethylsulfoxide (°C)|
In contrast to these results for DehRhb, the previous experiments with the thermostable DehSft enzyme  showed that activity was greatly reduced after exposure to different organic solvents for 1 h. DehSft lost more than 60% of its activity towards the substrate MCPA when incubated in 30% dimethylsulfoxide or 10% ethanol, 10% methanol or 10% acetonitrile. Other published studies with the Pseudomonas putida l-HAD have reported higher activity towards substrates with longer carbon-chain lengths, such as 2-bromohexadecanoic acid in the presence of anhydrous dimethylsulfoxide . The l-DEX YL enzyme also showed increased activity towards this substrate in n-heptane, although the activity towards substrates with short carbon-chain lengths, such as MCPA, decreased by 94% in comparison with untreated protein .
As industrial processes are often carried out in the presence of organic solvents it is advantageous to have a solvent-stable enzyme. The organic solvent can improve or change the substrate specificity and solubility . Both the thermal shift experiments in the presence of solvents and the solvent incubation assays demonstrated that DehRhb has a relatively high tolerance to solvents. Other studies with enzymes from halotolerant microorganisms have shown the proteins to have good stability in organic solvents [18, 31].
As determined from the thermal shift experiment (Fig. 3A), DehRhb has a melting temperature of 67 °C. The residual activity after incubation of DehRhb at temperatures of 30–70 °C showed that the enzyme retained 85% of its activity after incubation at 50 °C for 90 min (Fig. 3B). Enzyme activity was reduced after incubation of the enzyme at 60 °C, with the enzyme retaining just 14% activity after 90 min of incubation. The optimal temperature for the activity of the DehRhb is 55 °C.
Other l-HADs from both mesophilic and thermophilic sources have been biochemically characterized. DehSft from the thermophilic S. tokodaii shows high thermal stability, retaining 92% of activity after incubation at 50 °C for 90 min, and has a temperature optimum for activity of 60 °C . This is to be expected as S. tokodaii is a thermophilic archaeon isolated from the volcanic hot springs of Oita Prefecture in Kyushu, Japan . However, l-DEX YL, from the mesophilic Pseudomonas sp. YL, is also relatively thermostable, retaining 100% of activity when incubated at 60 °C for 30 min . The optimal temperature for activity of l-DEX YL is 65 °C despite Pseudomonas species having an optimal growth temperature of 30 °C.
Thermophilic enzymes have been exploited in industrial applications because increased thermostability results in a more robust catalyst with increased solvent stability and less susceptibility to proteolysis . High thermostability allows enzymes to operate at elevated temperatures where non-natural substrates are more soluble. The reported experiments show that DehRhb is a relatively thermostable enzyme from a mesophilic marine bacterium from the Rhodobacteraceae family.
The DehRhb crystals grew from microbatch crystallization screens at 18 °C with equal volumes of protein and precipitant solution containing 0.2 m LiCl, 0.1 m Tris/HCl (pH 8.0) and 20% PEG 6000. Substrate soaks were conducted at pH 4.5 for 1 h, which proved to be sufficient for ensuring high occupancy in the active site.
Quality of the models
The native structure, sulfate complex, reaction intermediate structures with MCAA and MCPA and the substrate-bound structure with l-2-monochlorobutyric acid (MCBA) were refined to a resolution of ≥ 2.0 Å. For each model, the final round of refinement resulted in an Rfree value equivalent of 20.4% or better. Some residues were excluded from the model when poor electron density was observed at the C and N termini of the protein (Table 2). The G-factors calculated for each model confirmed that the structures have good stereochemical properties . The Ramachandran plot for the native structure revealed that 94.0% of the residues were in the most favoured regions, with a further 5.4% in the additionally allowed regions and a single residue of each subunit lying in the disallowed region, with the other DehRhb complex structures having similar statistics. Glu21 was an outlier in the Ramachandran plot in all DehRhb structures; this residue is in the active-site pocket of DehRhb and is well defined in the electron density map. Density for the DehRhb active-site complexes with MCAA, MCPA and MCBA were confirmed by calculating Fo − Fc omit maps (Fig. 4A,B,C). The occupancies of the intermediates were estimated by ensuring the similarity of B-factors of the ligands to neighbouring residues. The final refinement statistics and validation results for all structures are shown in Table 2. Residues Pro132 and Pro158 of each subunit in all structures are in the cis-conformation. Many residue side chains were modelled with alternative conformations, and in some cases secondary positions of the main-chain oxygen were also modelled.
|Crystal||Native l-HAD||l-HAD complex with sulfate||l-HAD reaction intermediate with MCAA||l-HADs reaction intermediate with MCPA||l-HADs substrate complex with MCBA|
|Resolution (Å)||39.93–1.79 (1.83–1.79)||55.34–1.64 (1.68–1.64)||42.3–1.96 (2.01–1.96)||41.97–1.66 (1.71–1.66)||79–1.74 (1.79–1.74)|
|Cell dimensions (Å), P212121||a, b, c = 43.8, 68.3, 159.7||a, b, c = 42.8, 68.6, 283.3||a,b,c = 42.4, 68.4, 158.0||a, b, c = 42.8, 68.9, 157.2||a, b, c = 42.7, 68.6, 158.0|
|No. of protomers||2||4||2||2||2|
|Solvent content (%) VM (Å3 Da−1)||45.4 (2.2)||37.1 (1.9)||43.0 (2.2)||45 (2.2)||43.5 (2.1)|
|Completeness||99.8 (99.8)||99.9 (99.8)||99.9 (100)||99.9 (100)||99.9 (99.9)|
|(I)/ δ (I)||15.1 (2.8)||18.2 (3.4)||13.6 (2.9)||19.5 (3.7)||14.8 (2.1)|
|Rmerge † (%)||0.085 (0.728)||0.075 (0.83)||0.099 (0.6)||0.073 (0.67)||0.055 (0.398)|
|Overall R-factor (%)||15.7||16.3||15.9||16.3||16.2|
|Rfree (5.1% total data)||20.4||20.5||20.6||19.7||20.3|
|No. of residues modelled|| |
|No. of waters modelled||746||884||180||506||242|
|No of ligands modelled||0||4||2||2||2|
|Rmsd bond length (Å)||0.012 (0.022)||0.010 (0.022)||0.015 (0.022)||0.011 (0.022)||0.012 (0.022)|
|Rmsd bond angle (°)||1.32 (1.98)||1.3 (1.98)||1.49 (1.98)||1.3 (1.98)||1.3 (1.97)|
|Wilson B factor (Å2)||29.8||24||24.1||22.1||20.9|
|Average B factor|
|REFMAC RMS error (Å2)||0.1||0.1||0.15||0.1||0.1|
|Ramachandran analysis (% of residues)|
Overall structure of DehRhb
The approximate dimensions of the DehRhb monomer are 40 Å × 27 Å × 33 Å. The DehRhb monomer contains two domains (Fig. 5A). The core domain is formed by residues 14–17 and 119–232 and has a typical ‘Rossmann fold’, which consists of six parallel open twisted β-strands with a Richardson  topology of +1x, +1x, −3x, −1x, −1x surrounded by four helices and three 310 helices. The cap domain is made up of a four-helix bundle formed by residues 26–115. Approximately 37% of the residues in each subunit are in α-helices, 11% are in β-sheets and 5.5% are in 310 helices. The core and cap domains of DehRhb are structurally similar to those of DehIVa, DhlB, DehSft and l-DEX YL, with rmsd values between matching Cα positions of 1.41 Å over 195 residues, 1.41 Å over 194 residues, 1.40 Å over 178 residues and 1.41 Å over 200 residues, respectively. The cut-off distance of 3.5 Å was used to calculate these values. The active site is located between the cap and the core domain following the β1 strand. The side chains of amino-acid residues from both domains contribute to the active-site cavity.
The asymmetric unit of the native DehRhb crystal contains a biological homodimer, which is in agreement with the estimated size of the protein in solution, as determined by size-exclusion chromatography. The oligomeric state of DehRhb is consistent with those of DehIVa, DhlB and DehSft, which are also dimeric proteins. The two subunits of DehRhb are related by a molecular twofold axis to which the α2 helices from each subunit are parallel (Fig. 5B). The dimer has approximate dimensions of 70 Å × 40 Å × 43 Å. Upon the formation of the DehRhb dimer, 18.7% of the surface of each monomer is buried, in comparison with 13.4%, 13.5% and 19% in DehSft, l-DEX YL and DhlB, respectively.
The DehRhb dimer is similar to the dimers of other HADs, with the subunit interface formed by the α2 and α3 helices. The main differences between the different enzymes regarding this interface are the conformations and lengths of the loops. In DehRhb these loops are made up of residues 61–64 and 79–84 from the cap domain, which are located at the N- and C-termini of helix α3, and the loop formed from residues 201–213, which belong to the core domain. There are other areas of contact between the two subunits. The α2 helix interacts with α2 and α8 helices from the other subunit. The loop between β5 and β6 is in close proximity to the α3 helix from the adjacent subunit. These interactions, which are involved in dimer formation in DehRhb, are different from the dimerization interactions previously described for l-DEX YL  and DehSft . However, Tyr53 in DehRhb and the equivalent residues in DehIVa (Tyr57), DehSft (Tyr31), l-DEX YL (Tyr57) and DhlB (Tyr45) are conserved and involved in dimer stabilization.
The native, substrate and intermediate complex structures are quite similar with an rmsd value of ≤0.55 Å between Cα atom positions of the individual monomers. There appears to be no conformational changes of the DehRhb after substrate binding or the formation of reaction intermediates.
Native, sulfate, intermediate complex and substrate-bound forms of DehRhb
The structure of DehRhb has been solved in five different forms by altering the composition of the ‘mother liquors’, in which crystals were soaked before freezing for data collection. Native crystals were grown at pH 8.0 and soaked in a cryoprotectant at pH 8.0. To obtain the reaction intermediate complex structures, the pH of the ‘mother liquor’ was lowered to pH 4.5, as the reaction intermediate cannot be hydrolysed below pH 5.0 [12, 13]. Initial soaks with substrates at pH 4.5 did not result in ester intermediate complexes, as the sulfate ion present in the buffer competes for binding in the enzyme active site at low pH. The sulfate–DehRhb complex showed clear electron density for a tetrahedral-shaped sulfate ion in the active site next to the catalytic Asp18 residue in all four chains. The sulfate ion is coordinated by residues Asp18, Thr124, His183 and Lys157.
Further crystal-soaking experiments were conducted in a sulfate-free, Tris/HCl ‘mother liquor’ at pH 4.5 in an attempt to obtain the reaction intermediate complex structures. Clear continuous electron densities were observed for the ester intermediates of MCAA and MCPA bound to the catalytic Asp18 (Fig. 4A,B). The MCAA ester intermediate was modelled in the active site in two different conformations. The major and minor conformers have occupancy of 0.7 and 0.3 respectively, with the minor conformer in a position similar to that of the ester intermediate of MCPA. Crystal-soaking experiments were also conducted with the weak substrate MCBA at 25 mm and pH 4.5. The resulting complex did not contain a covalent intermediate, but instead the MCBA substrate was bound as a prereaction Michaelis complex at an occupancy of 0.6 in the active-site cavity (Fig. 4C). Two water molecules (observed in the same position in the native structure) were also modelled in the substrate carboxyl-binding site at an occupancy of 0.4.
Active site of DehRhb
The MCAA and MCPA intermediate complexes of DehRhb confirm Asp18 to be the main catalytic residue, which attacks the C2 of the substrate to form the ester intermediate in the first half of the reaction. The structure of the MCPA intermediate complex has the C2 atom in a D configuration, confirming that inversion of the chiral carbon occurs upon the halogen-removal step in the first part of the reaction . The intermediates of MCAA and MCPA and the substrate MCBA are located in a pocket with Asp18 on one side and a predominately hydrophobic aromatic environment on the other side, consisting of residues Phe47, Leu50, Ile51, Phe66, His183 and Trp185 (Fig. 4A,B). No movement of the main chain or side chains of residues are observed either upon substrate binding or during the first step of the enzymatic reaction.
Unlike the hydrolysis of the ester intermediate, the SN2 nucleophilic attack by Asp18 on the C2 of the substrate in the first step of the reaction can proceed at low pH. The lower activity of DehRhb towards long-chain substrates is confirmed by observing the MCBA bound in the active site as a Michaelis complex rather than the intermediate ester complex after a 1-h soak. This is in contrast to the MCAA and MCPA substrates, which form the high-occupancy ester complexes after a 1-h soak at pH 4.5.
For successful dehalogenation of the substrate, the halogen should be located in the hydrophobic cradle, with the C2-halogen bond aligned in a straight line with OD1 of Asp18 (12). The additional carbon atom (C4) of the MCBA shows a sterical clash with active-site residues, reducing the probability of MCBA being in a favourable orientation for nucleophilic attack by Asp18, thus rendering MCBA a weak substrate. The carboxyl group of MCBA occupies the same site as the carboxyl group of the MCPA reaction intermediate and the minor conformer of the MCAA reaction intermediate.
The ester bond in the reaction intermediate occupies an identical position in both the MCAA and MCPA complex structures, with the OD1 oxygen forming an H-bond with His183, as observed for the Asp18 in the native structure. The carboxyl group of the major conformation of the MCAA intermediate makes contact with the side-chain nitrogen atoms of Asn125 and Lys157 and the main-chain nitrogen of Asn125. It should be noted that the major conformation of the MCAA complex of DehRhb has not been observed in other known complexes of l-HADs.
The carboxyl group of MCPA makes H-bonds with the main-chain nitrogens of Val19, Asn20, Asn125 and the OG1 of Thr124. This group is also shifted nearly 2 Å deeper into the active site, and is rotated 90o in comparison with the MCAA major conformation. This conformation is similar to that previously observed for the intermediate ester complexes in other l-HADs. Manual positioning of the MCPA ester intermediate in a conformation similar to that observed for the major conformer of MCAA would result in the substrate C3 atom occupying the position of the catalytic water, which would prevent hydrolysis of the ester intermediate complex. The MCAA intermediate does not have a C3 atom and can accept an additional conformation, which forms fewer H-bonds than the MCPA intermediate. The hydrolysis of the MCAA intermediate seems to be able to proceed in both conformations, with the major conformation not bound as tightly, resulting in a faster release of the product. If the hydrolysis of the MCAA was possible only in minor conformation, the reaction rate for this substrate would drop below that of the MCPA, which was not observed. The higher energy of the MCPA binding may slow down the product-release step of the enzymatic reaction, explaining the lower activity observed for this substrate compared with MCAA. It is also possible that the extra carbon in the MCPA substrate could sterically hinder the initial binding of this substrate to the active site of DehRhb. This could slow down the first step of the reaction, also contributing to the different rates of activity observed with the two substrates.
Some dehalogenase structures have a clearly defined free halogen ion-binding site. For example, the crystal structure of Sphingomonas japonicum UT26 haloalkane dehalogenase has a chloride ion bound at a high occupancy in the active site . However, no halide-binding sites have been observed in the crystal structures of most HAD structures. The exception reported so far is the structure of the DhlB enzyme, which in the presence of 20 mm MCPA or MCAA was found to contain a chloride ion bound in close proximity to the catalytic intermediate. The chloride ion is coordinated by Arg139 and Asn115 and sits in a hydrophobic pocket formed by the side chains of Leu43, Phe58 and Phe175. This halide position maps a halide-stabilizing cradle, which is necessary for positioning of the substrate C2-halogen bond in line with the attacking OD1 of the active site aspartate before cleavage of this bond . No halide site was observed in the DhlB structure soaked with the same substrates at pH 8.0. This suggests that the halogen ion does not have high affinity for the site and is probably trapped upon formation of the intermediate complex at low pH.
Although the crystallization media contained 100 mm sodium chloride, no bound halogen ions could be located in any of the DehRhb structures. A DehRhb crystal was soaked in 1 m sodium bromide at pH 8.0 and data were collected close to the bromine absorption peak wavelength. Anomalous difference maps calculated with refined model phases (data not shown) did not show any features in the catalytic cavity, confirming the low affinity of a free halogen ion in the active site of DehRhb.
The MCBA halogen group is thought to map the halogen cradle in DehRhb, which is formed by the side chains of Phe47, Ile51, Phe66, Asn125 and Trp185. As a result of steric clashes of this substrate in the active site, the halogen group of MCBA could be slightly misplaced from the reaction position. Interestingly, the side chain of Trp185 has an alternative conformation in subunit A of the MCBA complex structure, indicating the possibility of some induced change in shape of the halogen cradle.
Structural features affecting substrate specificity
In order to interpret the enzyme's stereospecificity, both MCPA and d-2-chloropropionic acid were modelled in the active site of DehRhb as substrates before formation of the ester intermediate. The substrates were modelled with the carboxyl group in the same orientation as in the MCPA intermediate structure, with the C2-Cl bond pointing away from OD1 of Asp18. In positions where the chloride of the substrate did not overlap with neighbouring residues, the C3 atom of the modelled d-2-chloropropionic acid clashed with the main-chain oxygen of Asn20. This steric restriction prevents activity of the enzyme towards d-haloacids. The MCPA model fits the active site in a position similar to that observed for l-lactate in the active site of DehSft (Protein Data Bank code 2w11) [14, 36]. The orientation of the C2-Cl bond in the modelled MCPA prereaction complex is 40° different from that observed in the MCBA Michaelis complex structure.
Similarly to many other l-HADS, DehRhb has higher activity towards brominated substrates in comparison with chlorinated substrates (Fig. 1). This is expected as bromide is a better leaving group. Some exceptions exist, such as DehSft, which has slightly higher activity towards MCPA than towards l-2-bromopropionic acid . This appears to be the result of steric restrictions imposed by the narrow active site in this enzyme where the halide-binding residue, Arg23, is shifted towards the active-site aspartate, favouring binding of the smaller MCPA substrate.
The DhlB enzyme shows highest activity with dibromoacetic acid and dichloroacetic acid, in comparison with MCPA and MCAA . The wider active site of DhlB accommodates the second nonhydrolysable halogen of dihaloacetic acid, but is too large for the C3 carbon of MCPA. Therefore, when dihaloacetic acid is bound in the active site of DhlB, its C2-halogen bond is fixed in a position favourable for the nucleophilic attack by the catalytic aspartate. In comparison, when MCPA and MCAA bind to the active site of DhlB, this bond is not fixed in one position, which slows down the reaction. Similarly, the C3 atom of MCPA is fixed in the narrow active site of DehSft, which favours high activity towards MCPA. Mobility of the MCAA substrate slows down the reaction in DehSft, which also seems to have lower affinity for bulkier dihalogenated haloacids . The l-DEX YL enzyme, which prefers substrates with longer chains, has a significantly more open active site than other haloacid dehalogenases. DehRhb has the highest activity towards monobromoacetic acid as it has a more constrained active site, which does not have a favourable site for the C3 of MCPA or the nonreactive halogen of dihalogenated haloacids.
Structural comparisons and the DehRhb mechanism
The DehIVa enzyme shares 22% sequence identity with DehRhb. Point mutations of the DehIVa active-site residues Asp11, Arg42, Ser119, Lys152, Ser176, Asn178 Asp181 and equivalent residues in l-DEX YL resulted in a loss of activity of between 70 and 100% [37, 38]. These residues are conserved in previously characterized l-HADs. However, some of these residues are not present in the DehRhb enzyme (Fig. 5C). The most interesting changes are in the active site of DehRhb where conserved residues Arg42, Asn178 and Gly14 found in DehIVa are replaced with Phe47, His183 and Glu21 in DehRhb (Fig. 6A).
The residues Arg42 and Asn178 in DehIVa have been proposed to be involved in a ‘lock down’ mechanism of the substrate . This was based on the observation of different conformations of Arg42 between the empty and intermediate-bound active sites. In the case of the DehRhb enzyme where the equivalent arginine and asparagine residues are missing, this ‘lock down’ mechanism cannot be responsible for substrate binding. This may explain the lack of movement of residue side chains observed in the DehRhb active site upon complex formation compared with the ‘open’ and ‘closed’ positions seen in DehIVa. The conserved Arg42 is the positively charged residue in the halogen cradle in DehIVa. Its absence in DehRhb may slow down the halogen-removal step in this enzyme, which does not have charged residues involved in halogen binding.
When the catalytic water molecule was observed in a constrained environment in the active site of DehIVa , Asp181 was proposed to participate in the activation of the catalytic water molecule required for hydrolysis of the ester intermediate . Molecular dynamics calculations with the l-DEX YL model  concluded that the equivalent Asp180 and Lys151 (Asp186 and Lys157 in DehRhb) are responsible for deprotonation of the catalytic water molecule in the second part of the enzyme reaction (Fig. 6B).
In DehRhb, the catalytic water molecule is coordinated by His183 and the conserved Asp186. This residue makes an ion pair with the conserved Lys157 which is H-bonded to OD1 of the MCPA intermediate. The His183 makes an H-bond with the catalytic Asp18 on one side and an ion pair with Glu21 (an outlier on the Ramachandran plot) on the other side (Fig. 6C). This forms a catalytic triad similar to that observed in the haloalkane dehalogenases .
In all of the DehRhb structures, the catalytic water molecule is positioned for potential deprotonation by His183. The water is close to the plane of the imidazole group of this histidine and to the potential hydrogen atom position of the ND1 atom (Fig. 6C). In the DehRhb enzyme the catalytic water is far from the plane of the carboxylate group of Asp186, which makes water deprotonation by this residue unlikely.
In the DehlVa structure, the catalytic water is lying in a position convenient for deprotonation by the corresponding Asp181, in the plane of its carboxylate group (Fig. 6B). When the structures of the propionate intermediate complexes of DehIVa and DehRhb are superimposed, the corresponding Asp181 and Asp186 have similar orientations. However, the catalytic water in DehRhb is shifted by ∼1.5 Å away from the plane of the aspartate carboxylate group (Fig. 6A). We therefore propose that for the DehRhb enzyme, the catalytic water is activated by the His183-Glu21 dyad (Fig. 7) and not by the conserved Asp186-Lys157 pair, as in previously characterized l-HADs.
DehRhb from a marine bacterium in the Rhodobacteraceae family shows highest activity towards brominated substrates with short carbon chains (≤C3). The enzyme shows a high tolerance to solvents and is stable for 90 min at temperatures of up to 50 °C, with an optimal activity at 55 °C.
The DehRhb, that is both solvent and thermally stable, has the potential to be used for industrial applications in the pharmaceutical sector for the production of optically pure drug intermediates, including hydroxyalkanoic and haloalkanoic acids. The DehRhb enzyme has kinetic reaction values comparable with those of other characterized l-HADs.
The crystal structures of the complexes of DehRhb formed with covalently bound substrates MCAA and MCPA have confirmed Asp18 as the main catalytic residue in this enzyme. Residues His183 and Glu21 in the active site of DehRhb are proposed to form a dyad, which activates the catalytic water for attack of the ester intermediate in the second part of the dehalogenase reaction. The structure of the Michaelis complex for DehRhb with the longer-chain substrate MCBA shows that there is a steric clash of this substrate C4 atom within the active site. This prevents it from occupying a favourable position for dehalogenation, which explains why it is a poor substrate.
The different structures of DehRhb reported in this paper have provided an insight into the mechanism of this marine l-HAD enzyme, which appears to be novel with respect to the active-site residues involved in water activation. In DehRhb the catalytic water molecule appears to be activated by a His/Glu dyad, which is not present in other l-HADs. The DehRhb enzyme therefore represents a new member within the l-HAD enzyme family and shows features that have not been previously observed in related l-HAD enzymes.
Material and methods
Gene identification, cloning and overexpression
A bacterium from the Rhodobacteraceae family was isolated from a polychaeta worm collected from Tralee beach, Argyll, UK, which tested positive for l-HAD activity towards the substrate MCPA. The Rhodobacteraceae genome was sequenced using an Illumina GA2 sequencer. Using the ncbi blast tool  on a Galaxy bioinformatics pipeline , a single l-HAD gene (DehRhb) was identified within the 4.5 Mbp genome. The DehRhb sequence has been deposited in the GenBank databank with the accession number JX868516.
The DehRhb gene was cloned from genomic DNA into the pET-28a multicopy plasmid with the incorporation of an N-terminal His-tag. The DehRhb protein was overexpressed in BL21-CodonPlus (DE3)-RIPL E. coli and the recombinant protein was purified by nickel affinity chromatography and gel-filtration chromatography on a Superdex 75 column. The purified protein was stored in 0.1 m Tris/H2SO4 (pH 8.2) containing 0.1 m NaCl (buffer C). The purity of the recombinant DehRhb was analysed using SDS/PAGE .
l-HAD activity was measured using a previously described colorimetric assay  with some modifications. The assay solution had final concentrations of 1 mm Hepes, 1 mm EDTA, 20 mm sodium sulfate, 10 mm substrate and 20 μg·mL−1 of phenol red (pH 8.2). To initiate the assay, 180 μL of dehalogenase assay solution was mixed with 20 μL of purified protein (0.2 mg·mL−1 in buffer C). Unless stated otherwise all reactions were carried out at 25 °C with MCAA as the substrate. The reaction was followed by measuring a decrease in absorbance at 540 nm. To produce a standard curve, the standard dehalogenase assay solution was mixed with 1 m HCl to final concentrations of 0–2 mm in a total volume of 200 μL. As the temperature was found to have a slight effect on the activity of the assay, standard curves were performed at 10 °C intervals from 25 to 65 °C.
Activity assay experiments
The optimal temperature of the enzyme was determined by performing the assay at temperatures between 25 and 65 °C. The assay solution was preincubated at the set temperatures for 5 min before addition of enzyme.
The thermostability of the enzyme was determined by incubating the enzyme at temperatures between 30 and 60 °C for 30, 60 and 90 min, followed by incubation on ice for 10 min. The solvent stability of the enzyme was investigated by incubating the enzyme with ethanol, methanol, acetonitrile and dimethylsulfoxide at concentrations between 10% and 70% for 1 h at room temperature. The substrate specificity was determined by performing the standard assay with a range of substrates of varying chain lengths, halides and halide positions. Enzyme kinetic parameters were determined by carrying out the standard dehalogenase assay with bromoacetic acid at 55 °C. The substrate concentration ranged from 0.1 to 5 mm. The program GraFit version 6  was used to calculate the Km and Vmax values.
Thermal shift experiments
Thermal shift experiments were performed with a StepOne™ Real-time PCR system and the results were analysed using the protein thermal shift™ software, version 1.0 (Applied Biosystems, UK). The final mixture consisted of 20 μL of 0.075 mg·mL−1 of protein (in buffer C), 5 mm K2HPO4/KH2PO4 pH 7.5 and 4 × SYPRO Orange dye (Invitrogen). Solvent-stability experiments were performed in the same conditions with the addition of 0–50% methanol, ethanol, dimethylsulfoxide or acetonitrile.
The purified DehRhb was concentrated using a 10-kDa membrane (Vivaspin 20; Vivascience, Massachusetts, USA) at 3000 g and 4 °C until a final concentration of 10 mg·mL−1 was reached. The concentrated protein was subjected to microbatch crystallization screens at 18 °C. All crystals were grown in equal volumes of protein and precipitant solution containing 0.2 m LiCl, 0.1 m Tris/HCl and 20% PEG 6000 (pH 8.0). Before data collection, all crystals were soaked in a suitable ‘mother liquor’.
The native crystal was soaked in a ‘mother liquor’ containing 0.2 m LiCl, 0.1 m Tris/H2SO4, 18% PEG 6000 and 25% PEG 400 at pH 8.0 for 5 min. The sulfate complex crystal was soaked in mother liquor containing 0.2 m LiCl, 0.1 m Tris/H2SO4, 18% PEG 6000, 25% PEG 400 and 25 mm MCPA at pH 4.5 for 1 h. The crystals used for the intermediate complexes were soaked in a mother liquor containing 0.2 m LiCl, 0.1 m Tris/HCl, 18% PEG 6000, 25% PEG 400 and 25 mm substrate (MCAA or MCPA) at pH 4.5 for 1 h. The MCBA substrate-bound structure crystal was soaked in a ‘mother liquor’ containing 0.2 m LiCl, 0.1 m Tris/HCl, 18% PEG 6000, 25% PEG 400 and 25 mm MCBA at pH 4.5 for 1 h.
Data collection and structure determination
All diffraction data were collected at Diamond Light Source synchrotron (the beamlines are listed in Table 1). Data were processed with XDS  using the Xia2  pipeline. All further data and model manipulation were carried out using the CCP4 suite of programs .
A modified dimeric polyalanine DehIVa (22% sequence identity; Protein Data Bank code 2NO4)  model without the N and C termini (residues 1–8 and 202–225) was used for molecular replacement with the program MOLREP . The rotation function was calculated using a radius of 21 Å at a resolution of 2.5 Å and the translation search was performed at a resolution of 5 Å. Refinement of the resulting solution with REFMAC5  was initially unsuccessful. The model correction feature of MOLREP was used in which the model was modified according to the target sequence. This modified model was aligned to the previously found molecular replacement solution and was successfully refined. The solution was then submitted to automated refinement using arp/warp version 7.0.1 . The model was manually rebuilt in coot  and refined using REFMAC5.
Further structure complexes were solved using the final structure of the native DehRhb as a model for molecular replacement and model building, and refinements were conducted in the same manner as the native structure.
The Rfree statistical value using 5% of randomly selected data was used throughout refinement for all data sets. Dictionary definitions for the reaction intermediate and substrate-bound complexes were built using JLigand . The quality of the structures was checked using the program procheck . The final refinement statistics and validation results for the native DehRhb and complex structures are shown in Table 1. Structure factors and coordinates of the final models have been deposited in the Protein Data Bank with the following accession IDs: 2yml, 2ymm, 2ymp, 2ymq and 2yn4 for the native, sulfate-bound, MCAA and MCPA intermediate complexes and the MCBA substrate-bound structure, respectively. The program PyMOL  was used to produce Figures 4, 5A,B and 7.
The Biotechnology and Biological Science Research Council, UK and Aquapharm Biodiscovery, Oban, are acknowledged for PhD studentship funding (HRN). The authors thank the Diamond Light Source, UK, for access to beamline I03 (proposal number MX6851) and the beamline staff scientists. CS acknowledges a PhD GTA bursary from the University of Exeter. Funding in JAL's laboratory has been supported by the Wellcome Trust, BBSRC and EPSRC.