Elsevier

Process Biochemistry

Volume 92, May 2020, Pages 437-446
Process Biochemistry

Review
Haloacid dehalogenases of Rhizobium sp. and related enzymes: Catalytic properties and mechanistic analysis

https://doi.org/10.1016/j.procbio.2020.02.002Get rights and content

Highlights

  • Haloacid dehalogenases catalyse dehalogenation by two fundamental mechanisms.

  • They involve hydrolytic liberation of halide directly or through ester intermediates.

  • In L-DEXs, dehalogenation proceeds by SN2 nucleophilic attack conferred by Asp.

  • Dehalogenation of enantiomeric compounds invert the configurations of the product.

Abstract

Haloacid dehalogenases catalyse the cleavage of carbon − halogen bonds in halogenated organic acids. These enzymes are of high interest due to their potential applications in bioremediation and in synthesis of various industrial products. The efficiency of dehalogenases in various applications can be enhanced, provided that their molecular catalytic mechanisms are fully understood. Herein, we review the current understanding of enzymatic haloacid dehalogenation mechanisms and the important amino acid residues that are necessary for the enzyme’s catalysis, with special emphasis on haloacid dehalogenases produced by Rhizobium sp.

Introduction

Halogenated organic compounds are ubiquitous in biosphere. The number of these compounds is alarmingly increasing, from fewer than 50 naturally produced compounds in the year 1968 to more than 5000 in 2015 and still on the increase [1,2]. Naturally produced halogenated organic compounds are either biogenically synthesised by organisms or formed geogenically during abiotic processes in environment, such as forest fires, volcanic eruptions and other geothermal processes [3]. Halogenated organic compounds do not only occur naturally, diverse use of halogen based compounds in various industrially related products, such as solvents, agrochemicals and pharmaceuticals introduce extra anthropogenic halogenated organic compounds into the environment [4]. These compounds have caused serious environmental pollution and health problems owing to their direct toxicity, their potentially toxic breakdown products and their persistence in the environment [5].

Halogenated organic compounds are mineralised/detoxified by removal of the halide ion, which is responsible for the toxic and recalcitrant character of the compounds. Various studies have reported the potential of various chemical and biological methods in remediating polluted environment caused by halogenated organic compounds [[6], [7], [8], [9]]. Chemical dehalogenation usually involved the use of chemical reagent and often require a combustion step. Example of such process is the used of sodium naphthalene, which react with the halogenated pollutant to produce sodium halide and a neutral sludge that is subsequently incinerated [7]. Although this process is safe, it is very complex as it requires an air-free reaction vessel, thus limiting its application to industries. Other chemical methods have been developed as alternative to this and similar methods, such as oxidative dehalogenation. The major disadvantage of this method is that, a certain high temperature must be attained before the reaction can occur. This challenge is bridge in the more recent chemical methods that employ the use of catalyst. This has been demonstrated in the chemical dehalogenation of aqueous environment using hydrogen gas in presence of palladium catalyst on carbon substrate [7]. However, this method is only applicable to water environments. In general, no single chemical dehalogenation process is ideal, as they are often associated with one demerit or another.

Fortunately, a number of microbes use halogenated organic compounds as their sole source of carbon and energy, thereby helping to reverse the effects of environmental halogen-associated pollution. This process plays an important role in bioremediation of environment pollution due to natural or anthropogenic organohalides, thus forming the basis of the biological treatment of halogenated compounds. Application of bioremediation in treatment of halogenated organic compounds contaminated environment is generally preferred over the other approaches mainly because it is environmentally friendly [10]; since no toxic or foreign chemical is introduced into the environment. In the process, the native environmental organisms degrade the toxic pollutant into environmentally benign molecules which can also serve as energy source for other organisms, thereby maintaining the natural habitat. One good example of organisms used in bioremediation of pollution due to halogenated organic compounds is Rhizobium sp. RC1, which is able to grow in presence of 2,2-dichropropionate and 2-chlopropionate [11]. This bacterium produces three different dehalogenase enzymes namely, DehD, DehL and DehE, which catalyse the cleavage of carbon–halogen bonds in halogenated organic compounds [12,13]. While DehL and DehD are stereospecific toward L- and D-isomers of 2-chloropropionate, respectively, DehE degrades both isomers of the stereospecific dehalogenases [[14], [15], [16]]. These facts raised a fascinating question as to why Rhizobium sp. RC1 produces DehL and DehD since DehE can do the work of the both enzymes. Whatever might be the reason, the ability to produce these three dehalogenases affords the Rhizobium sp. RC1 ability to degrade and or utilise wide range of halogenated organic pollutants, thus makes it an important candidate for molecular engineering to produce enhanced dehalogenases that can be applicable in bioremediation and in synthesis of chirally active industrial chemical intermediates. Furthermore, bioaugmentation is considered as the most common strategy through which bioreaction is achieved. This in turn can be implemented mainly in three ways [17]. (1) Stimulation of the existing native environmental organisms that degrade the pollutant by addition of nutrients. (2) isolation of the degrading organisms from the polluted environment, followed by laboratory selection and returning of the most adapted strains back to the polluted environment, or (3) Genetic engineering of the native degrading organisms to improve its efficiency before reintroduction to the environment. Being soil the normal habitat of Rhizobium sp. RC1, makes it an ideal candidate for application in the last two aforementioned strategies, as it naturally adapted to grow in such environment.

However, to rationally engineer an enzyme, full molecular details of its catalytic mechanism and knowledge of the potential amino acid residues that upon mutagenesis would influence its catalytic efficiency and enantioselectivity are required. This review critically analyses the catalytic and mechanistic properties of dehalogenases of Rhizobium sp. RC1. and related dehalogenases from other bacteria. In addition, the possible reasons why Rhizobium sp. RC1 produces three dehalogenases are discussed.

In 1979, Berry and colleagues isolated from soil a bacterium capable of utilising 2,2-dichloropropionate as its sole source of carbon and energy, which they tentatively identified as a fast growing species of Rhizobium [18]. The bacterium growth can also be supported by 2-chloropropionate but not by monochloroacetate, dichloroacetate or 3-chloropropionate [18]. The ability to utilise these halogenated compounds is associated with the dehalogenase enzymes the bacterium produces, which catalyse the release of the chloride ions from these compounds. Gel electrophoresis analysis of the DNA extracts from the bacterium revealed the presence of not less than two dehalogenases with relatively broad substrate specificities [11]. The same study showed monohalogenated acetates to be susceptible for one of the dehalogenases, yet potent inhibitors for the other.

Dehalogenases from Rhizobium sp. were genetically characterised using a series of mutant strains [12]. The Rhizobium sp. RC1 mutant strain produced by chemical mutagenesis lost its ability to utilise 2,2-dichloropropionate or D, L-2-chloropropionate as its sole carbon and energy sources. Three secondary mutants were isolated after culturing the original mutant strain on 2,2-dichloropropionate and/or D, L-2-chloropropionate-containing agar. In the presence of 2,2-dichloropropionate, two secondary mutants, type 1 and 2 were recovered. The type 1 reverted to the wild type phenotype (revertant), for which all three dehalogenase (i.e. 2,2-dichloropropionate, D and L-2-chloropropionate) activities could be induced. The type 2 mutant constitutively produced DehE, which attacks 2,2-dichloropropionate, D- and L-2-chloropropionate, monochloroacetate and dichloroacetate. Selective utilisation of L-2-chloropropionate but not D-2-chloropropionate or vice versa were not observed under any of the tested conditions. The selective pressure induced by the presence of D, L-2-chloropropionate resulted to generation of type 3 mutant that constitutively produced DehL and DehD but could not produce DehE. DehL attacks only L-2-chloropropionate and dichloroacetate while DehD specifically liberate halide from D-2-chloropropionate and monochloroacetate [13]. All the three dehalogenases invert the enantiomeric configuration of their enantiomeric substrates after dehalogenation. The properties of these dehalogenases are summarised in Table 1.

The mutation sites in the original mutant strain have not been identified; however they were proposed to be within the regulator gene, which would affect production of the three dehalogenases provided that their genes are all controlled by this regulator [12]. Obtaining the type 1 revertant (with the wild type phenotype) requires a reversion of the original mutation in the regulator gene, or a repressor mutation in the regulator gene. Similarly, to produce the type 2 and 3 secondary mutants, separate mutations in the promoter regions controlling expression of DehE and DehD/DehL are required respectively (Fig. 1).

The genes encoding the three dehalogenases of the Rhizobium sp. RC1 have been sequenced, and the location of dehD was found to be 177 non-coding base pairs upstream of dehL (Fig. 1) [19]. Conversely, the location of dehE relative to that of the other two is not known. The deduced amino acid sequences of DehL and DehD are only 18 % identical, indicating that these dehalogenases do not have many common features [19]. This degree of sequence identity is similar to that found for Pseudomonas putida AJ1 HadD and HadL [20,21].

To date Rhizobium sp. RC1 remains the only bacterium that produces three different dehalogenases, DehD, DehE and DehL [22]. The substrate specificities of all the three dehalogenases and the degree to, which they liberate the halide ions were described by Huyop et al. in different studies. Analyses of halide ion assays showed that the stereospecific dehalogenases, DehL and DehD dehalogenate their respective isomers of L, D-2-chloropriopionate with catalytic efficiency value of 1.33 × 105 M―1s―1 [15] and 1.12 × 105 M―1s―1 [23] respectively. However, the non-stereospecific dehalogenase, DehE released halide from all substrates of the both stereospecific dehalogenases, but with ten-fold less catalytic efficiency than the stereospecific dehalogenases do [16]. These facts suggest that, while DehE can catalyse the release of halide ions from all substrates of both DehL and DehD, the individual stereospecific dehalogenases are preferred by their respective enantiomeric substrates. Although no study report how the three dehalogenases evolved, the available data lead us to speculate that the non-stereospecific dehalogenase, DehE preceded the more specialised stereospecific dehalogenases, which evolved from the DehE over time due to adaptation. Also, possibly, not all the three dehalogenases are expressed at all conditions. Evolution studies that will aim at isolating and characterising dehalogenases of the Rhizobium sp. RC1 in L, and/or D-2-chloropriopionate environments will shade more light on why the bacterium produces the three dehalogenase and their expressions pattern.

Members of haloacid dehalogenase (HAD) superfamily belong to a large group with diverse substrate specificity, to which epoxide hydrolases, P-type ATPase and different types of phosphatases belong [24]. HADs catalyse the hydrolytic cleavage of carbon-halogen bond from α-halogen substituted carboxylic acids e.g. D, L-2-chloropropionate and chloroacetate. L-DEX from Pseudomonas sp. YL was the first haloacid dehalogenase for which the crystal structure was solved [25]. Since then the structure of other haloacid dehalogenases have become available [[26], [27], [28], [29], [30]]. Analysis of these structures have provided crucial insights into the functional mechanism of the HAD superfamily.

Generally, haloacid dehalogenases catalyse dehalogenation of aliphatic haloacids by two mechanisms. The first mechanism is a two-step process, in which either an aspartate or glutamate residue near the N-terminal involves in an SN2-type nucleophilic substitution with the halogen bearing carbon atom, to release the halide ion and form an ester intermediate (Fig. 2a). In the second step, a basic amino acid residue, either, a lysine or histidine residue activates the catalytic water, which hydrolyses the ester intermediate to release the products [31,32]. Other catalytically important residue is arginine, which is involved in halide binding that stabilises the substrate and facilitates the halide ion abstraction. This mechanism is only evident among L-2-haloacid dehalogenases and fluoroacetate dehalogenases [[31], [32], [33]].

Alternatively, in the second model (Fig. 2b), a catalytic residue of the enzyme activates a water molecule, which directly attacks the α-carbon atom of the haloacid to substitute the halogen atom. This mechanism bypasses the ester intermediate formation and was first observed in D, L-haloacid dehalogenase from Pseudomonas sp. 113 [34]. Since then more haloacid dehalogenases that catalyses dehalogenation via similar mechanism were identified, which majorly constitute D-specific and D, L-haloacid dehalogenases [30,35].

The DehL of Rhizobium sp. RC1 is a member of L-2-haloacide dehalogenases (EC 3.8.1.2), the dehalogenase family that catalyses the specific conversion of L-isomer of 2-halocarboxylic acids to their corresponding D-2- hydroxycarboxylic acids [36]. This group is well characterised with few members having experimentally solved structures and fully established reaction mechanisms [26,27,35,37,38]. However, DehL structure has not been experimentally solved, it catalytic mechanism is described based on a homology model structure [39]. The structural modeling study reported that the three-dimensional structure of the DehL constitutes a combination of α-helices and β-sheets, which are organised in two domains to form the overall 3D structure [39]. The main domain, which is an α/β-type structure comprising of six central parallel β-sheets (β1–β6), alternated by five α-helices (α5–α9) in a right-handed β-α-β fashion. While the second domain called sub-domain, is a distorted four-helix-bundle structure fold-type, solely comprising of four antiparallel α-helices (α1–α4).

Analysis of the DehL three-dimensional structure provided more understanding into it functional and catalytic mechanisms. Inter-fragment interaction energy (IFIE) calculations between DehL and its typical substrate, L-2-chloropropionate revealed Asp13, Thr17, Arg51, Val18 and Met48 residues of DehL to be significantly interacting with the substrate directly (Fig. 3a) [40]. This suggests that these residues play essential roles in DehL catalysis. Being dehalogenation a hydrolytic reaction, a water molecule called catalytic water also partakes in the reaction. Analysis of molecular dynamics simulation results reported four water molecules within 5 Å of Asp13 in the DehL active site, which upon arrival of the L-2-chloropropionate, were forced to exit the active site [39]. Two different water molecules termed WT1 and WT2 were observed within the active sited when it was occupied by the L-2-chloropropionate, thus indicating one of the two water molecules may be the catalytic water. However, WT1 was confirmed as the catalytic water by IFIE calculations, in a more recent study [40]. The reported IFIE values between WT1 with Asp13, Thr17 and His184 were −23.75, 5.24 and −12.50 kcal/mol, respectively, while no significant interaction was observed between WT2 with any DehL residue (Fig. 3b).

Furthermore, the genuineness of the integrations between the DehL residues − Asp13, Thr17, Arg51, Val18 and Met48 with L-2-chloropropionate or WT1 identified by IFIE calculations, were validated by distance consistency between the interacting atoms over a short molecular dynamics simulations [40]. The distance between OD1 atom of Asp13 and Cα atom of L-2- chloropropionate was reported to be approximately stable around 4 Å, indicating the existence of an interaction between the two atoms that held them at relatively the same position. Similarly, the distances between interacting atoms of the other interacting residues − Thr17, His184, Gly15 and Gly16 with WT1 or L-2- chloropropionate or Asp13 atoms were found to be relatively stable within intractable distances. It is therefore on this basis, Adamu and colleagues [40] proposed the DehL dehalogenation catalytic mechanism.

In light of the enzyme reaction mechanism, L-2-haloacid dehalogenases have been described to generally catalyse the removal of halides by a two-step process [41,42]. The reaction proceeds via SN2 substitution mechanism with the carboxyl group of aspartate serving as the nucleophile to produce an ester intermediate. Subsequently, the ester intermediate is hydrolysed by an activated water molecule (catalytic water), thus allowing the release of the reaction products. Accordingly, the reported strong IFIE between Asp13 and L-2-chloropropionate; and the approximately constant distance between Asp13 OD1 and Cα atom of L-2-chloropropionate, strongly suggest that Asp13 plays a nucleophilic role in DehL dehalogenation mechanism [40] (Fig. 4). At this step, the well-positioned Asp13 OD1 interacts with L-2-chloropropionate Cα atom to release the chloride ion and form an ester intermediate. Structural analysis of DehL-L-2-chloropropionate complex suggested that the Asp13 OD1 atom is held at the position suitable for nucleophilic attack by the involvement of Asp13 carboxyl group in four hydrogen bonds network (Fig. 5), constituting two hydrogen bonds between Asp13 OD2 and each of the Gly15 H and Gly16 H atoms and another two between Asp13 OD1 and each of the Thr17 HG1 and Thr17 H atoms [39]. Notably, the nucleophilic role of Asp13 in DehL is consistent with the previously established fact that, the corresponding aspartate residue in L-DEX (Asp10) is conserved among members of haloacid dehalogenase (HAD) superfamily [43].

For the second (i.e. hydrolytic) step, structural analysis showed His184 and Arg51 to be the only basic residues within the DehL active site and proposed either of them may activate the catalytic water [39]. However, this was later investigated by fragment molecular orbital (FMO) calculations and molecular dynamics simulations [40]. The observed strong attractive IFIE between His 84 and WT1 and the insignificant IFIE between WT1 and Arg51 reported in the study, strongly suggest that His184 activates the catalytic water in DehL. Consistently, the constant distance between the His184 NE2 atom of His184 and WT1 OW during molecular dynamics simulation indicated His184 NE2 abstract the hydrogen atom from the catalytic water molecule. The highly electronegative hydroxyl group readily interacts with Asp13 side chain carboxyl carbon atom to hydrolyse the ester intermediate bond and to release the D-2-hydroxypropionate from the enzyme.

Recently, using quantum mechanics/molecular mechanics (QM/MM) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations, Adamu et al. have theoretically described the molecular basis of DehL enantiospecificity [44]. The study modeled the slow-reacting state (DehL-D-2-chloropiopinate complex) from the fast reacting-state (DehL-L-2-chloropiopinate complex) by substituent exchange mechanism. Various mutants of both complexes were generated by replacing the active site residues with other ones; and the corresponding binding affinity of the mutant and wild type DehL to both D, and L-2-chloropropionated were calculated and compared. The study found that mutation of Arg51 to Leu (R51 L) shutdown dehalogenation activity of DehL towards its natural substrate, L-2-chloropropionate; and on the other hand, M48R mutation afforded the enzyme a new activity towards D-2-chloropropionate. Combination of these two mutations in M48R|R51 L mutant was theoretically described to invert the DehL enantiospecificity by adjusting the charge and size of the substrate binding pockets to a configuration compatible to the opposite enantiomer of the natural substrate [44].

The mechanism of DehL catalysed dehalogenation is similar to that of L-DEX from Pseudomonas sp. YL as demonstrated by 18O incorporation experiments [31]. The experiments used multiple and single turnover reactions in H218O solvent using L-2-chloropropionate as substrate. The lactate produced by the multiple turnover reaction was labeled with 18O, whereas the lactate generated by single turnover reaction was not labeled. This indicates that the 18O was first incorporated into the enzyme from where it was then transferred to the reaction product. Tandem mass spectrometric analysis of the enzyme after multiple turnover reaction revealed that Asp10 is labeled with two 18O, indicating Asp10 acts as a nucleophile to attack Cα of substrates to for the ester intermediate. Asp105 in a fluoroacetate dehalogenase from Moraxella sp. B was also described to play the nucleophilic role in the enzyme dehalogenation [32]. Using molecular dynamic simulations and ab initio FMO calculations, Asp180 was shown to activate the catalytic water on its own or with Lys151, Ser175, and Asn177 in L-DEX [45], which subsequently hydrolyses the ester intermediate to release the hydroxylalkanoic acid from the enzyme.

The L-DEX catalysed dehalogenation monitored by mass spectrometry, structurally demonstrated the reaction path and confirmed the formation of the ester intermediate during the enzyme action [46]. When the L-DEX was incubated with L-2- chloropropionate, a peak corresponding to the native enzyme disappeared and a new one appeared, which later disappeared and then the original one reappeared. These strongly indicate, when the substrate was introduced, the enzyme was initially transformed to an ester intermediate, which was then hydrolysed to regenerate the native enzyme and products.

A site-directed mutagenesis study of L-DEX, which involved replacement of its highly conserved polar and charged residues with other residues, showed individual replacements of Asp10, Thr14, Arg41, Ser118, Lys151, Tyr157, Ser175, Asn177 and Asp180 with other residues, caused significant decrease in the enzyme dehalogenase activity [42]. Because replacements of these residues cause no conformational changes detectable by spectrophotometry and gel filtration, the residues are considered essential for the enzyme catalysis and are conserved in almost all L-haloacid dehalogenases [[47], [48], [49]]. Although, DehL catalyses dehalogenation by similar mechanism as the other L-haloacid dehalogenases, of the nine residues that when mutated negatively affect catalysis of L-DEX and are conserved among L-2-haloacid dehalogenases, only Asp10 is conserved in DehL. The corresponding active site residues in DehL are perhaps appropriately maintaining the functions conferred by the non-conserved residues, thus conserving the overall functional mechanism. The catalytic amino acid residues of DehL were identified by in silico alanine scanning experiments [40]. With the exception of Gly16 and Ala185, all the putative DehL active site residues were individually mutated to an alanine. The calculated absolute free binding energies of DehL-L-2-chloropropionate mutant complexes containing alanine substituted for Asp13, Thr17, Met48, Arg51, or His184, significantly decreased as compared to that of the wild type. All these residues were demonstrated by ab initio FMO calculations to substantially interact with the L-2-chloropropionate and/or catalytic water molecule, indicating their essentiality in DehL catalysis [40].

As a member of D-2-haloacid dehalogenase (D-DEX) family; DehD from Rhizobium sp. RC1 catalyses the hydrolytic cleavage of carbon − halogen bonds specifically in D-isomer of 2-haloacids to produce the corresponding L-2-hydroxyacids. D-2-haloacid dehalogenases are the least-studied group of haloacid dehalogenases. Until recently when the structure of HadD AJ1 from Pseudomonas putida AJ1/23 was crystallised [30], no structure of any D-2-haloacid dehalogenase is experimentally solved. However, before then, the catalytic mechanism of DehD has been predicted based on a homology model [50]. Molecular docking analysis of the natural substrate of DehD, D-2-chloropropionate into its active site revealed residue − Arg107, Arg134 and Tyr135 to interact with the substrate and assumed to be important in the enzyme catalysis. Because Arg134 is conserved among D- and D, L-haloacid dehalogenases, it was proposed that DehD catalyses dehalogenation in a similar way as DehI from Pseudomonas putida PP3 [51]. It was proposed that Arg134 activates the catalytic water molecule that subsequently attacks the Cα of the D-2-haloacid to release the halide ion and form the product without the formation of ester intermediate. However, this proposition is very unlikely since the water was shown to be activated by the oxygen atom of backbone carboxylic group of Arg134, which can be conferred by any residue.

The recently solved crystal structure of HadD AJ1 provide detailed and more reliable insights into the molecular mechanism of D-2-haloacid dehalogenases catalysed dehalogenation [30]. Co-crystallization and crystal soaking of HadD AJ1 with either reactive or nonreactive substrates yielded no enzyme-substrate complex crystal; instead an enzyme-product complex was captured at 2.18 Å resolution. Contrary to L-2-haloacid dehalogenases, these suggest that HadD AJ1 catalyses dehalogenation without the formation of ester intermediate.

Further examination of the enzyme-product complex structure revealed the active site of HadD AJ1 constitutes 71 Å3 enclosed binding pocket lined by thirteen amino acid residues. Comparison of HadD AJ1 active site residues with those of D,L-DEXs showed Trp48, Asn131, Tyr134, Ser204 and Asp205 reissues to be highly conserved. In another enzyme-product complex, a water molecule adjacent to the product was observed to be trapped in the HadD AJ1 active site. The water molecule is about 3 Å away from the Cα atom of the product, and it interacts with Asp205 and Asn131 through hydrogen bonds. Because Asp189 in DehI, which corresponds to the conserved Asp205 of HadD AJ1 activates the catalytic water [28], Asp205 and Asn131 were proposed to play similar role (Fig. 6). This was confirmed in a site-directed mutagenesis experiment, where substitution of Asp205 with Asn leads to complete shutdown of the dehalogenation activity [30]. Similarly, replacement of Asn131 with Asp results to about 95 % loss of dehalogenation activity. Being the water molecule only 3 Å away from the Cα atom of the product, a distance favorable for an SN2 nucleophilic attack, provides grounds to hypothesise that HadD AJ1 catalyses dehalogenation hydrolytically. This hypothesis was tested by a single turnover reaction for HadD AJ1 and D-2-chloropropionate in H218O solvent. 18O-labelled L-lactate constituted about 80 % of the product generated, confirming that the activated water molecule was actually the molecule that attacks the Cα atom of the substrate to release the halogen and subsequently produce the product.

Unlike DehL and DehD that attack specific stereoisomers of enantiomeric haloacids, DehE of Rhizobium sp. RC1 non-stereospecifically removes halogens from both D and L isomers of haloacids. Although the structure DehE has not been solved experimentally, its dehalogenation mechanism has been substantially studies based on a homology model. The amino acid sequence of DehE and that of DehI from Pseudomonas putida PP3 are 72 % identical, thus the two D, L-haloacid dehalogenases have similar structures [52,53]. Analysis of DehE-L-2-chloropropinates and DehE-D-2-chloropropinates complex structures obtained by molecular docking revealed the putative DehE active site is lined with twelve residues, Trp34, Ala36, Phe37, Asn114, Tyr117 Ala187, Ser188, Asp189, Tyr265, Phe268, Ile269, and Ile272 [53]. All these residues are conserved in DehI and D, L-DEX 113 from Pseudomonas sp 113 and therefore, are proposed to catalyse dehalogenation via the similar mechanisms. The amino acid residues that play crucial roles in DehE catalysed dehalogenation were determined by site directed mutagenesis experiments [54]. With the exception of the two alanine residues, all the putative DehE active site residues were substituted with different residues. Notably, replacement of Asp189 with Asn totally turned off DehE dehalogenation activity. Since Asp189 is conserved in DehI, and the corresponding residue was shown to be implicated in activation of catalytic water [28], Asp189 was proposed to play similar role in DehE. Additionally, individual mutations of W34A, F37A, and S188A drastically reduced DehE dehalogenation activity by not less than 80 % relative to that of the wild type, suggesting the relevance of these residues in DehE catalysis.

In general, all D, L-haloacid dehalogenases including DehE, DehI and D, L-DEX 113 catalyse dehalogenation by the same mechanism, which is also similar to that of D-DEXs (Fig. 6), for single reason that they have identical active site residues. The reaction mechanism was characterised based on the 18O incorporation experiments and site directed mutagenesis in L-DEX 113 [34]. Single turnover reaction using excess amount of L-DEX 113 in H218O solvent with either L- or D-2-chloropropionate substrate showed majority of the resulting lactate were labeled with 18O. This obviously indicates the solvent water was directly incorporated into the product molecule, contrasting the observation of similar experiments for L-DEX whose mechanism involves the formation an ester intermediate. To confirm the 18O from the solvent was not incorporated into the D, L-DEX 113 before it was being transferred to the lactate product, as in the case of L-DEX, 18O should not be detected in the enzyme. Accordingly, liquid chromatography/mass spectrometry analysis of the peptide fragments of the D, L-DEX 113 formed by digesting the enzyme after multiple turnover reactions in H218O solvent reveals no significant difference in molecular masses of all the peptides and the predicted ones for both D- or L-2-chloropropionate substrates. These also suggest the enzyme catalyses the dehalogenation of both D- and L-enantiomers via the same mechanism.

DehI is the first D, L-DEX for which its molecular structure is experimentally solved [28]. The crystal structure provides more details into the molecular mechanism of the entire enzyme group. Investigation of DehI active site interactions with D and L-2-chloropropionate substrates predicted by in silico molecular docking suggested three possible binding sites for methyl and chloride groups of the ligands, called site 1, 2 and 3. The carboxyl groups for both D and L- substrate isomers are placed at positions of the two sulfate oxygen atoms observed in the crystal structure, owing to the propensity of the electronegative oxygen atoms to bind the sulfate binding site and forming hydrogen bonds with Ser188 and Phe37. Strikingly, the most favourable binding modes for D and L- substrate isomers based on the GLIDE docking program scoring function, only differ in the binding site for chloride atom. The predicted distance between the OD1 atom of Asp189 and Asn114 with the Cα in the both substrate isomers is approximately 4.7 Å. This coupled with the fact that when water molecule was docked into DehI active site in the absence of the substrate, it binds adjacent to same Asp189 and Asn114 residues at appropriate position for nucleophilic attack, indicate their involvement in the catalytic water activation. This is also consistent with reported mutation of Asp194 in D,L-DEX 113 (corresponding to Asp189 in DehI) to Asn resulted to total shutdown of activity [55].

The proposed mechanism is that the side chain amino group of Asn114 interacts with and stabilises the Asp189 by forming hydrogen bond with the Asp189 OD2 atom. This positioned the Asp 189 OD1 at a position appropriate to attack the catalytic water molecule, thereby activating it. The activate hydroxyl molecule, acting as a nucleophile attacks the Cα of the substrate to release the chloride ion, thereby forming the hydroxyacid product.

In nature, there exist a broad range of enzyme diversity that provides numerous opportunities for various desired functions such as bioremediation and industrial biocatalysis. However, most of these enzymes are not primarily directed toward those functions, they are rather coincidental or ancillary function. Advances in biotechnology and more understanding of protein structure and function allow us to modify existing proteins in a way that they can perform our desired functions.

Redesigning enzyme is a multifaceted process that can be approached differently, depending on the target property for which the enzyme is to be engineered. Since engineering of dehalogenase enzymes is majorly aimed at increased substrate utility and stereospecificity, its corresponding function is generally repurposed by rational approach [44,56]. This approach is well demonstrated in a study that investigated the molecular basis of DehL stereospecificity [44]. Using various computational techniques, the study theoretically inverted the enzyme stereospecificity and identified the specific residues responsible for that function (Fig. 7). The enzyme engineering process begins by computational modeling of all possible target reaction complex conformations (slow-reacting complex), DehL- D-2CP from the fast-reacting complex, DehL- L-2CP. A total of four DehL-D-2CP conformations were generated; and subsequently screened by QM/MM optimisation and based on the displacement of chiral center to yield only one most probable candidate. The active site of enzyme of the resulting DehL-D-2CP complex was rationally engineered by rounds of in silico site-directed mutagenesis to generate mutant library, after which the mutants were screen for activity. The study observed that M48R and R51 L mutations afford the enzyme activity towards the opposite stereoisomer of its natural substrate, D-2CP, thus inverting the enzyme stereospecificity [44].

Generally, the success of protein engineering in enhancing catalytic activity and physical properties of enzymes cut across various classes of enzyme including dehalogenases. Enzyme engineering of a haloalkane dehalogenase (DhaA) from Rhodococcus rhodochrous improves its dehalogenation activity towards 1,2,3- trichloropropane by 32-fold compared to the wild type enzyme activity [57]. Combination of various rational design and directed evolution methods were used to obtain the improved DhaA mutant. Random acceleration molecular dynamics was employed to probe the ligand entry to and exit from the active site; and to identify hot spots for mutagenesis. Identified hot spots at the access tunnels were modified by site directed and site saturation mutagenesis to allow generation of exhaustive mutant library with all possible amino acid combinations at those points. The mutants with improved activities were screen and confirmed by the kinetic analyses. Sequencing and molecular dynamic simulations analyses revealed that the most enhanced DhaA mutant harbours I135 F, C176Y, V245 F, L246I and Y273 F mutations in the access tunnels connecting the buried active site with bulk solvent. Four of the five mutations in the enhanced mutant involved replacement of small amino acid with large aromatic residue, hence narrowing the access tunnels, thereby reducing the accessibility of the active site for water, which compete with the substrate for interaction with the nucleophile. This favours active complex formation and enhances the overall reaction.

Recently an attempt has been made to transfer stereospecificity by active site transplantation between two haloalkane dehalogenases [58]. DhaA from Rhodococcus rhodochrous and DbjA from Bradyrhizobium japonicum are both haloalkane dehalogenase with identical catalytic pentad and demonstrate similar stereospecificity toward α-bromoesters [59]. However, only DbjA demonstrates peculiar stereospecificity for β-bromoalkanes, which is attributable to it fragment 138-HHTEVAEEQDH-150 termed ERB fragment [60]. Therefore, the DbjA stereospecificity for β-bromoalkanes should in principle be transferred to DhaA by addition of the ERB fragment to the DhaA protein. It was against those backgrounds that Sykora and colleague transplanted the DbjA ERB fragment to the DhaA by cumulative site directed mutagenesis [58]. Although structural crystallography confirmed that the active site geometry of the mutant DhaA matched that of the DbjA, its stereospecificity not significantly altered. Molecular dynamic simulations and time-dependent fluorescence shifts showed that the dynamics and hydration at the entrance of the tunnel significantly vary between the designed and target enzymes, hence the reasoned why the stereospecificity is not transferred. These point out the importance of dynamics and hydration for enzymatic catalysis and design.

Rhizobium sp. RC1 produces three different haloacid dehalogenases namely, DehL, DehD and DehE. DehL and DehD are stereospecific and are only active toward L- or D-stereo form of enantiomeric haloacids. In contrast, DehE is non-specifically acts on both L- and D-haloacids isomers. Although none of the three dehalogenases from Rhizobium sp. RC1 has experimentally solved structure, their molecular mechanisms were extensively studied and described by homology models based on crystal structures of similar dehalogenases. DehL catalyses the removal of halide ion from haloacids via similar mechanism as do the other L-DEXs such as L-DEX from Pseudomonas species YL and HadL from Pseudomonas putida AJ1, by a two-step process. Firstly, a nucleophilic aspartate residue attacks the Cα atom of the substrate to release the halide ion and form ester intermediate. Secondly, a basic residue activates a catalytic water molecule, which then hydrolyses the ester intermediate and release the hydroxyacid product. However, the residues that influence DehL catalysis are different from those in the other L-DEXs. On the other hand, the mechanisms of DehD and DehE dehalogenation are the same, similar to those demonstrated in D, L-DEXs, which does not involve the formation of the ester intermediate seen in L-DEXs. In most cases, a water molecule activated by an aspartate residue directly attacks the Cα of the substrate to break the Cα−Halogen bond and releases the halide ion, thereby generating the hydroxyacid product.

The current advancement in computer sciences make it possible to predict molecular structure of protein computationally with remarkably acceptable precision, so long as it has at least a homologue whose molecular structure is experimentally solved. With high computing power and appropriate numerical algorithms, various computational methods can provide detailed description of biological systems, which are otherwise very difficult to obtain or even unachievable by experiment. Therefore, the current availability of crystal structure of at least one representative of dehalogenase group provide enormous opportunity to describe the molecular mechanism of any dehalogenase even without solving it molecular structure experimentally. This will facilitate the provision of necessary information for the enzyme engineering to improve efficiency.

Section snippets

Author contributions

AA, FH conceived and designed review. AA prepared the manuscript. All co-authors reviewed and approved the final manuscript.

Funding

The study is partially sponsored by Universiti Teknologi Malaysia under Visiting Researcher scheme. UTM, Vote number: Q.J090000.21A4.00D20.

Declaration of Competing Interest

The authors declare no conflict of interest.

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