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 Active Site and Tunnels . 1084 3 Function of HLDs . ............................................................ 1085 3.1 Catalytic Activity . 1085 3.2 Substrate 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 enzymes 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 solvent, 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-enzyme 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), enzyme kinetics (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-hydrolase 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 catalytic triad), 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-binding site, which is needed to stabilize the negatively charged transition state that occurs during hydrolysis (Verschueren et al., 1993c). This oxyanion hole 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). Catalysis 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 histidine. A catalytic acid stabilizes the charge developed on the imidazole 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),