Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of Its Entrance Tunnel

Abstract

Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using sitedirected mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.Haloalkane dehalogenases are microbial enzymes acting on haloorganic compounds. The enzymes cleaves the carbon-halogen bond and replaces a halogen with a hydroxyl group from a water molecule (1). Activity and specificity of haloalkane dehalogenases is not optimal for industrial applications (2), and numerous studies have been conducted to improve their catalytic properties using in vitro techniques (3-13). Engineered enzymes can be used in biotechnology applications, such as detoxification of environmental pollutants and bioorganic synthesis. Such technologies are already in use (14) or are under development (2, 15, 16). Furthermore, haloalkane dehalogenases has become an important model system for in silico study of molecular principles of enzymatic catalysis (12,(17)(18)(19)(20)(21)(22)(23)(24).Haloalkane dehalogenase LinB (25) is the enzyme isolated from a ␥-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimob...

Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

“…The substrate specificity of an enzyme is determined largely by the residues lining the active site and pathway to the active site. It has been demonstrated for other bacterial ␣/␤ hydrolases that the mutation of surface residues at the active-site tunnel entrance can alter substrate selectivity (8). By completely blocking access from one side of the active site, Cif may be restricting the majority of traditional epoxide substrates from being hydrolyzed.…”

Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

“…Access tunnel mutations have been shown to have substantial effect on HLD activity in other HLD members. 10,11 Comparison of DhlA to the other HLD-I structures identifies three key differences unique to DhlA: a 10 amino acid insertion in the loop comprising the entrance to the slot tunnel, the repositioning the side-chain torsion angle of the highly conserved halide-stabilizing Trp175, and the blocking of the main tunnel through the position of Trp194 in DhlA, all of which result in DhlA's restricted binding pocket unlike other HLD-I structures. The insertion has been cited as the key reason that DhlA has activity on chlorinated compounds.…”

“…The substrate specificity and activity of HLDs have been rationalized by comparing structural differences in the tunnel(s) that provide access to the active site from the surface of the enzyme. 10,11 Crystal structures of DhlA, DppA, and DmrA 18,19,22 overlay with an RMSD of 1.5 Å or less. However, three key differences in DhlA distinguish it from DppA and DmrA: an insertion of 10 residues that reduces the size of the slot channel by folding into the channel entrance, a Trp blocking the main entrance tunnel, and a rotation of the halide-stabilizing Trp175.…”

“…The accessibility of the active site varies and it has been shown that the size and properties of the access tunnels are key determinants of substrate specificity and catalytic efficiency of the enzymes. 10,11 The five catalytic residues common to all HLDs comprise a nucleophile (Asp), general base (His), and catalytic acid (Asp or Glu), plus a pair of halide-stabilizing residues (Trp, Tyr, or Asn). 12 The composition and precise positioning of this "catalytic pentad" varies among three phylogenetically-derived HLD subfamilies: Asp-His-Asp 1 Trp-Trp for HLD-I, Asp-His-Glu 1 Asn-Trp for HLD-II, and Asp-HisAsp 1 Asn-Trp for HLD-III.…”

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae

“…The HLD active site is built up of an entrance tunnel and a hydrophobic pocket for substrate binding [37]. The residues lining the substrate pocket mainly belong to the cap domain and define substrate specificity.…”