Key Residues for Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution

Abstract

bHalohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and ␤-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E S ] ‫؍‬ 1.7 to R enantioselectivity [E R ] ‫؍‬ 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E S > 200) and a 5-to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.

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

“…Strikingly, in HheA the aromatic side chain of the Senantiomer of the substrate was docked in the pocket formed by residues Leu141, Asn144, and Asn178, among which Leu141 and Asn178 are at the equivalent positions of Trp139 and Asn176 in HheC. By manipulating these active-site residues, a variant with an inverted enantioselectivity (from E S of 21.7 to E R of 13) and an S-selective HheA variant toward 2-CPE have been developed (Asn178Ala) (26). The results indicated that the two enzymes might share similar mechanisms for controlling enantioselectivity.…”

“…Kinetic parameters of the wildtype HheC and its variants toward 1,3-DCP and both enantiomers of 2-CPE were taken in 50 mM Tris-SO 4 buffer (pH 8.0) at 37°C by monitoring halide liberation using purified enzymes as described before (26,34).…”

“…So far, only one S-selective mutant of halohydrin dehalogenase from Arthrobacter AD2 (HheA) has been evolved by using a semirational design approach, which can be used together with the R-selective HheC to produce optically pure halohydrins and epoxides in an enantioconvergent manner (26). However, the S-selective HheA mutant showed a very low catalytic efficiency in the dehalogenation of halohydrins.…”

Stereoselective Chemoenzymatic Synthesis of Optically Active Aryl-Substituted Oxygen-Containing Heterocycles

“…Some general examples of stereoselective syntheses of halohydrins, as precursors of optically active cyclic ethers, are (a) the reduction of halogen-substituted ketones by means of hydrides complexed with chiral ligands (e.g., CBS-catalyst) [20,21]; (b) stereoselective hydrogenation processes run in the presence of Rh/Ru catalysts [22][23][24]; (c) microbial [25][26][27][28] or isolated enzymes-mediated [29] stereoselective reductions of α-halo-acetophenones; and (d) the kinetic resolution of racemic mixtures using dehalogenases (e.g., HheC from Agrobacterium radiobacter AD1) [30,31]. Our group recently focused on the development of new bio-catalyzed whole-cell biotransformations for the enantioselective preparation of chiral secondary alcohols, which are valuable precursor compounds for active pharmaceutic ingredients (APIs) [32][33][34][35].…”

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

“…For this purpose, a viable approach employs rational and random protein engineering strategies that can yield drastically improved and functionally diverse enzyme variants (28). Such strategies have been successfully applied to HheA2 (29)(30)(31) and HheC (13,(32)(33)(34), addressing specific drawbacks of the respective parental enzymes and yielding HHDH mutants with sometimes substantial improvements with regard to target reactions. Nevertheless, parental sequences will always govern the overall accessible sequence space in every protein engineering study.…”