Biocatalytic and Structural Properties of a Highly Engineered Halohydrin Dehalogenase

Abstract

Two highly engineered halohydrin dehalogenase variants were characterized in terms of their performance in dehalogenation and epoxide cyanolysis reactions. Both enzyme variants outperformed the wild-type enzyme in the cyanolysis of ethyl (S)-3,4-epoxybutyrate, a conversion yielding ethyl (R)-4-cyano-3-hydroxybutyrate, an important chiral building block for statin synthesis. One of the enzyme variants, HheC2360, displayed catalytic rates for this cyanolysis reaction enhanced up to tenfold. Furthermore, the enantioselectivity of this variant was the opposite of that of the wild-type enzyme, both for dehalogenation and for cyanolysis reactions. The 37-fold mutant HheC2360 showed an increase in thermal stability of 8 °C relative to the wild-type enzyme. Crystal structures of this enzyme were elucidated with chloride and ethyl (S)-3,4-epoxybutyrate or with ethyl (R)-4-cyano-3-hydroxybutyrate bound in the active site. The observed increase in temperature stability was explained in terms of a substantial increase in buried surface area relative to the wild-type HheC, together with enhanced interfacial interactions between the subunits that form the tetramer. The structures also revealed that the substrate binding pocket was modified both by substitutions and by backbone movements in loops surrounding the active site. The observed changes in the mutant structures are partly governed by coupled mutations, some of which are necessary to remove steric clashes or to allow backbone movements to occur. The importance of interactions between substitutions suggests that efficient directed evolution strategies should allow for compensating and synergistic mutations during library design.

Recent Progress in the Synthesis of Super-Statins

“…Schallmey et al [101] reported the detailed biochemical characterization of two highly engineered halohydrin dehalogenases that can transform (S)-4-chloro-3-hydroxybutyrate 17 to (R)-4-cyano-3-hydroxybutyrate 19, via (S)-3,4-epoxybutyrate 18. Both of these variants of halohydrin dehalogenase were evaluated in dehalogenation and epoxide cyanolysis reactions on various substrates, and both of them outperformed the wild-type enzyme in the cyanolysis of 18 to 19.…”

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining

“…Reaction mixtures were incubated at 30°C, and 50-l samples were taken after 5, 20, and 60 min of incubation to monitor dehalogenase activity by using the halide release assay as described elsewhere (35). In addition, after 60 min, the remaining 650 l of the reaction mixture was extracted once with 600 l methyl tert-butyl ether containing 0.1% (vol/vol) dodecane as an internal standard.…”

“…To assess whether these recombinant enzymes possess true HHDH activity, a colorimetric activity assay for halide release from chloroalcohols 1,3-dichloro-2-propanol and 2-chlorophenylethanol, as well as from the bromoalcohol 1,3-dibromo-2-propanol, was performed with extracts of cells containing the recombinantly expressed enzymes (35). Afterwards, gas chromatography analysis was used to confirm the formation of the corresponding epoxides.…”

“…For example, HheC has been shown to be extraordinarily tolerant, with mutations at 153 of its total 254 residues; a maximum of 42 simultaneous substitutions per variant have been described (13). However, these heavily engineered enzyme variants are still rather similar to parental HheC; for example, HheC-2360 (35) shows more than 85% sequence identity. Clearly, novel sequences would be a valuable addition to the functional diversity of the HHDH enzyme toolbox.…”

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

“…The enzymes which are involved in the biodegradation pathway of halopropanols catalyze the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing epoxide and HCl. Halohydrin dehalogenase HheC has been studied extensively because of its high enzymatic activity and high R-enantioselectivity toward some short-chain aliphatic and aromatic substrates (14)(15)(16)(17). The enzyme has been applied in the production of optically pure epoxides and most of the R-enantiomers of ␤-substituted alcohols through kinetic resolution, with a maximum yield of 50% (18)(19)(20).…”