20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases J. Damborsky* . R. Chaloupkova . M. Pavlova . E. Chovancova . J. Brezovsky Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic *[email protected]

1 Introduction ...... 1082

2 Structure of HLDs ...... 1083 2.1 Catalytic Residues ...... 1083 2.2 and Tunnels ...... 1084

3 Function of HLDs ...... 1085 3.1 Catalytic Activity ...... 1085 3.2 Specificity ...... 1086

4 Engineering of HLDs ...... 1086 4.1 Mutants with Modified Activity ...... 1086 4.2 Mutants with Modified Thermostability ...... 1090 4.3 Mutants with Modified Substrate Specificity ...... 1090 4.4 Mutants with Modified Enantioselectivity ...... 1092 4.5 Research Needs ...... 1093

K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-540-77587-4_76, # Springer-Verlag Berlin Heidelberg, 2010 1082 20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases

Abstract: The structure–function relationships for haloalkane dehalogenases, representing one of the best characterized families of involved in degradation of halogenated compounds is described. A substantial amount of mechanistic and structural information is currently available on haloalkane dehalogenases, providing good theoretical framework for their modification by protein engineering. Examples of constructed mutants include variants with modified: (1) activity, (2) thermostability, (3) substrate specificity and (4) enantioselec- tivity. Some variants carried mutations in the tunnels, connecting the buried active site with surrounding , rather in the active site itself. Mutagenesis in the residues lining the protein tunnels represents a new paradigm in protein engineering.

1 Introduction

Haloalkane dehalogenases (HLDs, EC 3.8.1.5) are bacterial enzymes cleaving a carbon–halogen bond in halogenated hydrocarbons. The very first HLD was isolated from Xanthobacter autotrophicus GJ10 in 1985 (Keuning et al., 1985) and served as a paradigm for carbon– halogen bond cleavage in halogenated aliphatic hydrocarbons. Since then, a number of newly isolated and biochemically characterized HLDs grown to 14 enzymes. HLDs have been isolated from bacteria colonizing contaminated environments (Janssen et al., 1988; Keuning et al., 1985; Kumari et al., 2002; Nagata et al., 1997; Poelarends et al., 1998; Poelarends et al., 1999; Sallis et al., 1990; Scholtz et al., 1987; Yokota et al., 1987), but interestingly also from pathogenic organisms (Jesenska et al., 2000; Jesenska et al., 2002; Jesenska et al., 2005). Phylogenetic analysis revealed that the HLD family can be divided into three subfamilies denoted HLD-I, HLD-II and HLD-III, of which HLD-I and HLD-III are predicted to be sister groups (Chovancova et al., 2007). A substantial amount of mechanistic and structural information is currently available on HLDs. The unique tertiary structures were determined by protein crystallography for DhlA, isolated from X. autotrophicus GJ10 (Franken et al., 1991), DhaA from Rhodococcus sp. TDTM0003 (Newman et al., 1999), LinB from Sphingo- bium japonicum UT26 (Marek et al., 2000), DmbA from Mycobacterium tuberculosis H37Rv (Mazumdar et al., 2008) and DbjA from Bradyrhizobium japonicum USDA110 (Prokop et al., 2009), whereas more then thirty crystal structures of protein-ligand complexes of HLDs are available in the Protein Data Bank (Supplementary Table S1). The structure and reac- tion mechanism of HLDs (> Fig. 1) has been studied in detail by using protein crystallography (Liu et al., 2007; Marek et al., 2000; Mazumdar et al., 2008; Newman et al., 1999; Oakley et al.,

. Figure 1 General scheme of the reaction mechanism of HLDs. Alkyl- intermediate is formed in the first reaction step by nucleophilic attack of carboxylate oxygen of an aspartate group on the carbon atom of the substrate. This intermediate is in the second reaction step hydrolyzed by an activated water molecule, yielding a halide ion, a proton, and an alcohol as the products. Enz – enzyme. Structure–Function Relationships and Engineering of Haloalkane Dehalogenases 20 1083

2002; Oakley et al., 2004; Ridder et al., 1999; Streltsov et al., 2003; Verschueren et al., 1993a,b,c), site-directed mutagenesis (Pries et al., 1995a,b; Bohac et al., 2002; Chaloupkova et al., 2003; Hynkova et al., 1999; Krooshof et al., 1997; Pavlova et al., 2007; Schanstra et al., 1997; Schindler et al., 1999), (Bosma et al., 2003; Prokop et al., 2003; Schanstra and Janssen, 1996; Schanstra et al., 1996a,b) and molecular modeling (Banas et al., 2006; Bohac et al., 2002; Damborsky et al., 1997a,b; Damborsky et al., 1998; Damborsky et al., 2003; Devi-Kesavan and Gao, 2003; Hur et al., 2003; Kahn and Bruice, 2003; Kmunicek et al., 2001; Kmunicek et al., 2003; Kmunicek et al., 2005; Lau et al., 2000; Lightstone et al., 1998; Maulitz et al., 1997; Nam et al., 2004; Negri et al., 2007; Olsson and Warshel, 2004; Otyepka and Damborsky, 2002; Otyepka et al., 2008; Shurki et al., 2002; Silberstein et al., 2003; Soriano et al., 2003; Soriano et al., 2005). The number of practical applications employing HLDs are increasing with growing knowledge of their properties and structure–function relationships. HLDs can find their use in the bioremediation of environmental pollutants (Stucki and Thuer, 1995), biosensing of toxic chemicals (Campbell et al., 2006), industrial biocatalysis (Janssen, 2007; Prokop et al., 2004; Swanson, 1999), decontamination of warfare agents (Prokop et al., 2005; Prokop et al., 2006), as well as cell imaging and protein analysis (Los and Wood, 2007).

2 Structure of HLDs

HLDs structurally belong to the a/b- superfamily (Nardini and Dijkstra, 1999; Ollis et al., 1992). The proteins in this superfamily do not possess obvious sequence similarity, even though they have diverged from a common ancestor. The three-dimensional structure of HLDs is composed of two domains: (i) the a/b-hydrolase main domain, strictly conserved in various members of the a/b-hydrolase superfamily and (ii) the helical cap domain, variable in terms of number and the arrangement of secondary elements (> Fig. 2). The a/b-hydrolase fold is made mostly up of an eight-stranded parallel b-sheet which is flanked by a-helices and serves as a scaffold for the catalytic residues (Verschueren et al., 1993c). The cap domain is composed of several helices connected by loops. The cap domain is inserted to the main domain after the b-strand 6 and determines the substrate specificity (Kmunicek et al., 2001; Pries et al., 1994).

2.1 Catalytic Residues

The catalytic residues of HLDs always constitute a catalytic pentad: a nucleophile, a base, a catalytic acid (together a ), and a pair of halide-stabilizing residues (> Fig. 2). The composition of the catalytic pentad is not conserved among different subfamilies: Asp- His-Asp + Trp-Trp in subfamily HLD-I, Asp-His-Glu + Asn-Trp in subfamily HLD-II and Asp-His-Asp + Asn-Trp in subfamily HLD-III (Chovancova et al., 2007). The nucleophile is always located on a very sharp turn, known as the nucleophile elbow, where it can be easily approached by the substrate and the catalytic water molecule. The geometry of the nucleophile elbow also contributes to the formation of the oxyanion-, which is needed to stabilize the negatively charged transition state that occurs during hydrolysis (Verschueren et al., 1993c). This is formed by two backbone nitrogen atoms: the first is from the residue directly next to the nucleophile, while the second is located between strand b3 and 1084 20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases

. Figure 2 Molecular topology (a) and tertiary structure (b) of HLDs. a/b-hydrolase fold domain (white) and the specificity-determining cap domain (black) are distinguished. A nucleophile, a base and the first halide-stabilizing residue are conserved (filled symbols), whereas the catalytic acid and the second halide-stabilizing residue are variable among HLDs (empty symbols).

helix a1(> Fig. 2). proceeds by the nucleophilic attack of the carboxylate oxygen of an aspartate group on the carbon atom of the substrate, yielding displacement of the halogen as a halide, and by formation of a covalent alkyl-enzyme intermediate (> Fig. 1). The alkyl- enzyme intermediate is subsequently hydrolyzed by a water molecule that is activated by a . A catalytic acid stabilizes the charge developed on the ring of the histidine during the hydrolytic half reaction.

2.2 Active Site and Tunnels

The active site of HLDs is either a hydrophobic cavity (in DhlA) or hydrophobic pocket (in DhaA, LinB, DmbA, and DbjA) located at the interface of the main domain and the cap domain. The only polar groups localized in the active sites of HLDs are the residues of the catalytic triad. The active sites of HLDs differ in their size and accessibility to the solvent (> Fig. 3). The active site pockets can have as much as four times difference in volume: DhlA < ˙ DhlA < LinB < DmbA < DbjA. The active site cavity of DhlA is deeply buried in the protein ˙ core with limited accessibility to water molecules through a very narrow tunnel (Verschueren et al., 1993a), the active site pockets of DhaA and LinB are more accessible via the main tunnel and the slot tunnel (Petrek et al., 2006), while the pockets of DmbA and DbjA are the most exposed to solvent via the wide main tunnel and the slot tunnel (Prokop et al., 2008). These tunnels connect a hydrophobic active site with surrounding solvent and represent a very important structural feature of HLDs (Marek et al., 2000). The size, shape, physico-chemical properties, and dynamics of the tunnels are one of the determinants of activity and substrate specificity in HLDs. Tunnels play an important role during the following steps of the catalytic cycle: (1) binding of a substrate, (2) binding of catalytic water, (3) release of a halide ion, and (4) the release of an alcohol. The tunnels in HLDs can be either permanent or ligand-induced Structure–Function Relationships and Engineering of Haloalkane Dehalogenases 20 1085

. Figure 3 Anatomy of the active sites and tunnels in HLDs. The position of buried active site and two tunnels within protein structure is schematized in (a), where (1) denotes an active site, (2) denotes a main tunnel, and (3) denotes a slot tunnel. A surface of the active site and the tunnels is represented by wire in DhlA (b), DhaA, (c) LinB (d), DmbA (e), and DbjA (f).

(Klvana et al., 2009). The permanent tunnels are observable in the ligand-free crystal struc- tures, while the ligand-induced tunnels are only seen in the crystal structures of the protein- ligand complexes and in molecular dynamic trajectories. The solvation and desolvation of the active sites of HLDs through these tunnels is a very dynamical process due to high flexibility of the cap domain (Negri et al., 2007; Otyepka and Damborsky, 2002).

3 Function of HLDs

3.1 Catalytic Activity

A comparison of the kinetic mechanism of DhlA (Schanstra et al., 1996a), DhaA (Bosma et al., 2003) and LinB (Prokop et al., 2003) determined by transient kinetics reveals overall similarity (> Scheme 1; > Table 1). The binding of the substrate and the cleavage of the carbon–halogen bond are fast steps, resulting in the accumulation of the alkyl-enzyme intermediate for all three enzymes. The main and the very important difference in kinetic mechanism is in the rate- limiting step. The halide release is the predominant rate-limiting step for dehalogenation of 1,2-dichloroethane and 1,2-dibromoethane by DhlA (Schanstra and Janssen, 1996), liberation of alcohol for dehalogenation of 1,3-dibromopropane by DhaA (Bosma et al., 2003) and hydrolysis of the alkyl-enzyme intermediate for dehalogenation of 1-chlorohexane and bromocyclohexane by LinB (Prokop et al., 2003). The observation of different rate-limiting 1086 20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases

. Scheme 1 Kinetic mechanism of HLDs. E – enzyme, RX – substrate (halogenated alkane), E.RX – enzyme-substrate complex, E-R.X – alkyl-enzyme intermediate, E.X-.ROH – enzyme- complex, XÀ - halide

product, ROH – alcohol product. kx – kinetic constant of an individual catalytic step.

steps for three enzymes from the same demonstrates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolu- tionary closely related proteins.

3.2 Substrate Specificity

The HLDs are broad specificity enzymes. The set of substrates converted by HLDs consists of over a hundred chemical individuals – chlorinated, brominated and iodinated compounds; haloalkanes, haloalkenes, haloalcohols, halohydrins, haloethers, haloesters, haloacetamides, haloacetonitriles, and cyclohaloalkanes (Damborsky et al., 2001). Statistical analysis of sub- strate specificity profiles revealed the presence of several different specificity groups within this protein family (Damborsky et al., 1997c). Substrate specificity of HLDs is primarily deter- mined by the structure of the cap domain (> Table 2) and can be predicted from the statistical models employing three-dimensional structures of enzyme-substrate complexes. These com- plexes can be prepared by computer modeling and quantitatively analyzed by using multivari- ate data analysis (Kmunicek et al., 2003; Kmunicek et al., 2005). Analysis of four family members revealed that only a very limited fraction of the residues (<8%) contribute to the substrate binding and specificity, typically, explaining >85% of variance in Michaelis

constants Km. Van der Waals interactions with the residues of the first shell dominate substrate recognition in all studied HLDs (> Fig. 4). The residues of the tunnels contribute to substrate binding in LinB, DmbA, and DbjA, but not DhlA, due to low accessibility of the active site in DhlA (Brezovsky et al., unpublished).

4 Engineering of HLDs

4.1 Mutants with Modified Activity

1,2,3-trichloropropane (TCP) is a toxic non-natural compound released into the environment as a result of its manufacture, formulation, and use as a solvent and extractive agent. TCP has been detected in low concentrations in surface, drinking and ground water, with a half-life estimated to extend up to a hundred years under groundwater conditions (Yujing and Mellouki, 2001). TCP is very resistant to natural biodegradation under aerobic conditions. No natural strains, which are able to metabolize TCP have yet been isolated, opening the possibility for the construction of such a strain by genetic engineering. Construction of a dehalogenase enzyme with improved conversion of TCP is an essential step towards engineering a TCP-degrading strain. Bosma et al., (2002) applied DNA shuffling and error prone PCR on the dhaA gene to . Table 1 tutr–ucinRltosisadEgneigo aolaeDehalogenases Haloalkane of Engineering and Relationships Structure–Function Kinetic constants of DhlA, DhaA and LinB and their mutants. Rate-limiting steps are in bold

Enzyme Substrate KS k2 k-2 k3 k4 Km kcat kcat /Km À À À À À À À (mM) (s 1)(s1)(s1)(s1)(mM) (s 1)(mM 1.s 1) DhlA wt 1,2-dibromoethanea >27 >130 – 10 Æ 2 4 ± 1.5h 10 3 0.3 DhlA V226A 1,2-dibromoethaneb 110 60 Æ 20 – 12 ± 3 43 Æ 10h 33 8.2 0.25 DhlA F172W 1,2-dibromoethanec 63 30 Æ 5– 9 ± 1.5 75 Æ 25h 25 5.9 0.24 DhlA W175Y 1,2-dibromoethaned 250 70 Æ 15 – 8 ± 0.7 16 Æ 2h 60 5.8 0.0008 DhlA D260N + N148E 1,2-dibromoethanee 700 0.55 ± 0.05 – 0.8 Æ 0.1 >10h 430 0.35 0.0008 DhlA wt 1,2-dichloroethanea 2222 50 Æ 10 – 14 Æ 3 8±2h 530 3.3 0.0062 DhlA V226A 1,2-dichloroethaneb 5555 14 Æ 1– 9±2 50 Æ 10h 1500 3.8 0.0025 DhlA F172W 1,2-dichloroethanec 10000 4.5 ± 1 – 9.5 Æ 1 >75h 5130 2.9 0.0006 DhaA wt 1,3- 60–300 300 Æ 60 – 14.8 Æ 0.7 3.9 ± 0.6i 5 3.7 0.54 dibromopropanef LinB wt chlorocyclohexaneg >500 >40 – – – 221 0.1 0.0005 LinB wt bromocyclohexaneg >450 >200 1.1 Æ 0.4 2.5 ± 0.07 – 23 1.8 0.08 LinB wt 1-chlorohexaneg 240 Æ 44 117 Æ 5 0.4 Æ 1 3.2 ± 0.2 – 16 2.6 0.16 aDetermined at pH 8.2 and 30C (Schanstra et al., 1996a) bDetermined at pH 8.2 and 30C (Schanstra et al., 1997) cDetermined at pH 8.2 and 30C (Schanstra et al., 1996b) dDetermined at pH 8.2 and 30C (Krooshof et al., 1998) eDetermined at pH 8.2 and 30C (Krooshof et al., 1997) fDetermined at pH 9.4 and 30C (Bosma et al., 2003) gDetermined at pH 8.6 and 37C (Prokop et al., 2003) hHalide release 20 iAlcohol release 1087 1088 20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases

. Table 2 Structure-specificity relationships of HLDs with known tertiary structure

Active Enzyme Cap domain sitea Native substrate Preferred substrates DhlA 1 smallb terminally halogenated

DhaA 2.5 largec terminally halogenated vicinally halogenated b-halogenated

LinB 3 largec terminally halogenated vicinally halogenated b-halogenated cyclic

DmbA 3.5 Unknown largec monosubstituted terminally halogenated b-halogenated cyclic

DbjA 4 Unknown largec terminally halogenated vicinally halogenated b-halogenated b-methylated cyclic

aRelative volume bLength up to C3 cLength at least C6

improve the kinetic properties of DhaA for TCP conversion. The evolved dehalogenase mutant, C176Y + Y273F, was 3.5-times more active towards TCP than the wild type enzyme. Another variant of DhaA (Gray et al., 2001) also carried a substitution at position 176. This random variant of DhaA, G3D + C176F, was obtained by in vitro evolution and showed a 4-fold improvement in activity with TCP, relative to the wild type enzyme. Pavlova et al., (2009) combined advanced computer modeling with directed evolution and obtained twenty five unique protein variants with higher activities towards TCP than the wild type enzyme. The best mutant carried five single-point mutations and demonstrated 32-times higher tutr–ucinRltosisadEgneigo aolaeDehalogenases Haloalkane of Engineering and Relationships Structure–Function 20 . Figure 4 Interactions important for recognition of substrates by the active sites of HLDs: DhlA (a), LinB (b), DmbA (c), and DbjA (d). The interactions are labeled according to chemical character (van der Waals, v; electrostatics, e), ordered by their importance for the multivariate model and colored according to their localization in the first shell (blue), second shell (red) and tunnel (yellow). 1089 1090 20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases

activity (26-times higher catalytic efficiency) when compared to the natural wild type enzyme. The ‘‘hot spot’’ residues for saturated mutagenesis were selected by Random Acceleration Molecular Dynamics (Luedemann et al., 2000), simulating the release of the product from the enzyme active site. Interestingly, mutagenesis targeted the access tunnels rather than the active site. These tunnels connect the buried active site cavity with the surrounding solvent and the enhanced rate with TCP appears to be due to the absence of water molecules in the active site cavity promoting formation of an activated complex (> Fig. 5). Efficiently catalyzed reaction steps are followed by solvation of the active site by water molecules. Waters are attracted to the cavity from bulk solvent due to the presence of charged ion and assist release of products.

4.2 Mutants with Modified Thermostability

The HLDs represent a class of enzymes with a high potential for biocatalysis (Janssen, 2007). Performance of the biocatalytic process is a combination of the reaction rate of a biocatalyst and its stability. According to the Arrhenius relationships, the rate of the enzymatic reac- tion will approximately double for every 10C increase in temperature. Gray et al., (2001) attempted to improve stability of DhaA at higher temperatures to develop efficient biocatalytic process for the conversion of halogenated alkanes to halohydrin products (Swanson, 1999). They used a directed evolution technique called the Gene Site Saturation Mutagenesis (Kretz et al., 2004), which theoretically allowed all single site mutants to be sampled, in combination with high-throughput screening methods. Thermostability of parental dehalogenases and evolved mutants was measured by assaying activity at elevated temperatures. Eight single point mutations were discovered to be scattered along the protein sequence. This had consider- able effects on enzymes thermostability (> Fig. 6). A combination of all of these mutations yielded a variant D78G + F80S + T148L + G171Q + I209L + N227T + W240Y + P291A   with a 30,000-times longer half-life at 55 C, and an increase in Tm to 8 C. Stabilization of an a-helix by the mutation T148L, which is responsible for the formation of an addi- tional H-bond between the serine hydroxy group and an acceptor in F80S mutant, were important factors for the enhanced stability of enzymes. Effects of other mutations, includ- ing I209L and P291A, with the largest contribution towards improved thermostability were more difficult to explain. Three mutations N227T, W240Y, and P291A did not affect melting temperature, although did contribute to the increase of half life. A plausible mechanistic explanation is that these three mutations increase the possibility that the protein will refold more efficiently after denaturation. The complexity of the results demonstrate that our under- standing of structural basis of protein stabilization is still limited as it would be very difficult, if not impossible, to design these stabilizing mutations rationally.

4.3 Mutants with Modified Substrate Specificity

Investigation of the evolution of enzymes by selecting spontaneous mutants that convert a xenobiotic compound, represents a unique opportunity to observe how new substrate specificities evolve in Nature. 1,2-Dichloroethane (DCE) is a non-natural compound whose production and emission to the biosphere started in 1922. It is unlikely that sufficient selective pressure to evolve a complementary enzyme existed before this date (Pries et al., 1994) and the enzymes participating in the degradation of DCE must have undertaken recent evolutionary Structure–Function Relationships and Engineering of Haloalkane Dehalogenases 20 1091

. Figure 5 Water accessibility in the wild type DhaA (a) and its five-fold mutant I135F + C176Y + V245F + L246I + Y273F (b) with enhanced activity towards TCP. Spheres show the positions of water molecules inside the protein for snapshots collected during the molecular dynamics simulation. The spheres have a radius of 0.5 A˚ and are centered on the oxygen atoms of the water molecules. The mutants were designed by Random Expulsion Molecular Dynamics simulations and constructed by site-saturated mutagenesis (Pavlova et al., 2009).

. Figure 6 Substitutions accumulated in the eight-point mutant of DhaA with enhanced thermostability (D78G + F80S + T148L + G171Q + I209L + N227T + W240Y + P291A). All but one substitution (I209L) are located on the protein surface. The mutants were obtained by Gene Site Saturated Mutagenesis (Gray et al., 2001). adaptation. The first step in the utilization of DCE by soil bacterium X. autotrophicus GJ10 is catalyzed by DhlA, hydrolyzing this short-chain (C2) chloroalkane to the corresponding alcohol, which can further serve as source of carbon and energy for growth. In a fascinating laboratory evolution experiment, Pries et al., (1994) expressed DhlA in a strain of Pseudomo- nas that grows on long-chain (C6) alcohols and selected 12 independent mutants that utilize 1092 20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases

. Figure 7 Spontaneous substitutions (D170H and P168S), deletion (D164–174) and insertions (∇172–174, ∇152–153 and ∇145–154) localized in the cap domain of DhlA mutants with relaxed substrate specificity. The residues making the salt bridge D170-K261 between the main domain and the cap domain are shown in ball and stick. The mutants were obtained from an in vivo adaptation experiment (Pries et al., 1994).

1-chlorohexane. These mutants were obtained after 4 weeks in batch cultivations that contained a mixture of 1-chlorobutane and 1-chlorohexane as the sole carbon sources. Sequencing of evolved genes revealed six mutant dehalogenases with relaxed substrate specifi- cities: ∇145–154, ∇152–153, D164–174, P168S, D170H, and ∇172–174. Interestingly, none of the mutations directly affected the active site residues, with the exception of D164–174, in which the active site cavity forming residues F164 and F172 were missing. All observed mutations are located in a segment of the dhlA gene which encodes the N-terminal part of the cap domain (> Fig. 7). The mutants D164–174, P168S, D170H, and ∇172–174 carry changes that affect the structurally important salt bridge D170-K261. This salt bridge is positioned between two domains and its disruption will make the cap domain more floppy (Otyepka and Damborsky, 2002). The structural basis of the relaxed specificity in the other two mutants, ∇145–155 and ∇152–153, is more difficult to explain. The active site cavity could be enlarged due to insertions, but this is only speculation, as the residues surrounding the insertion are not in direct contact with the substrate of the wild type enzyme (Pries et al., 1994). These results present experimental evidence that the cap domain determines substrate specificity and that generation of the repeats is an important mutational event during its evolution. This evolutionary paradigm has been recently implemented into a novel directed evolution method which generates randomly repeats and deletions in vitro (Pikkemaat and Janssen, 2002).

4.4 Mutants with Modified Enantioselectivity

In response to the general awareness of the physiological and ecological advantages of the use of single enantiomers, the manufacture of enantiomerically pure compounds has become an expanding area of the fine chemicals industry. When pharmaceuticals, agrochemicals, food additives and their synthetic intermediates are marketed as single enantiomers, high Structure–Function Relationships and Engineering of Haloalkane Dehalogenases 20 1093 enantiomeric purities characterized by enantiomeric excess (e.e.) >98%, are required. Pieters et al., (2001) investigated chiral recognition of haloalkane dehalogenases DhlA and DhaA. The magnitude of chiral recognition was low; a maximum E-value of 9 could be reached after structural optimization of the substrate and the development of enantioselective dehalo- genases for use in industrial biocatalysis was defined as one of the major challenges of the field (Janssen, 2004). We have assayed DhaA, LinB, and DbjA for their enantioselective conversion of brominated and alkane substrates into chiral alcohols (Prokop et al., 2009). All three proteins possessed high enantioselectivity (>200) with a-brominated esters. DbjA additionally showed high enantioselectivity towards structurally simple molecule 2-bromopentane (E = 145), while DhaA and LinB showed only low enantioselectivity (E =7 and E = 16, respectively). Structural analysis revealed that DbjA contains a unique surface loop in its specificity-determining domain. Deletion of this loop has led to the mutant enzyme DbjAD with a significantly lowered enantioselectivity toward 2-bromopentane (E = 58). Enantioselectivity could be re-introduced by an additional single-point mutation DbjAD + H139A (E = 120). Introduced mutations modulated anatomy and water accessibility of the main tunnel in DbjA (> Fig. 8). A hydrophobic interaction of the alkyl chain with the wall of this tunnel accompanied by desolvation seemed to be important for enantioselective discrimination of the structurally simple molecule 2-bromopentane by DbjA. Another two studied family members, DhaA and LinB, do not have this water accessible cone-like tunnel and therefore cannot efficiently discriminate enantiomers of b-brominated alkanes. These results demonstrate that enantioselectivity of an enzyme can be modulated by engineer- ing of a protein tunnel via modification of a surface loop.

4.5 Research Needs

Isolation and biochemical characterization of new members of the HLD family continues to be of great interest. Characterization of new family members has led to new knowledge about structure–function relationships and the evolution of HLDs. These newly isolated enzymes,

. Figure 8 The deletion mutants of DbjA with modified tunnels and modulated enantioselectivity: wild type DbjA (a), D140–146 (b) and D140–146 + H139A (c). The region carrying deletion in the surface loop is shown in ribbon. The ‘‘gate-keeping’’ His/Ala139 are shown in stick. The mutants were designed based on sequence/structure comparisons and constructed by site-directed mutagenesis (Prokop et al., 2009). 1094 20 Structure–Function Relationships and Engineering of Haloalkane Dehalogenases

native or genetically modified, also hold great potential for practical applications which often require optimized properties: (1) high enantioselectivity with substrates that can be converted to valuable products by biocatalysis, (2) enhanced resistance to organic for decon- tamination purposes, (3) elevated activities with specific target compounds like TCP or DCE for bioremediation, (4) broadened pH-range for biosensors, and (5) increased thermostabil- ities and long-term stabilities for nearly every possible application. Development of better data management and tools for analysis are needed for mechanistic studies. The amount of data on HLDs, as well as many other enzymes, is growing exponen- tially and these tools will assist in the extraction of knowledge from this data. For example, we have only started to understand the importance of tunnels in HLDs for (de)solvation and the exchange of ligands between the active site and the surrounding environment, even though these processes are essential for function of proteins with buried active sites. The greatest challenge in the research of HLDs is the identification of their biological role. The genes coding for HLDs are widely distributed among various bacterial species, including the tissue-colonizing organisms, e.g., Mycobacterium tuberculosis or Mycobacterium bovis. The number of genes annotated as HLDs by sequence similarity in genomic and proteomic databases is growing. Though for many proteins encoded by these genes, dehalogenating activity has not yet been confirmed experimentally and their natural function in host organ- isms remains unknown.

Acknowledgments

Financial support of the Ministry of Education, Youth and Sports of the Czech Republic via LC06010 (J. Brezovsky, E. Chovancova, M. Pavlova) and MSM0021622412 (J. Damborsky, R. Chaloupkova) is gratefully acknowledged.

References

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