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

Abstract

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and ␤-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/ Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E R of 65 [WT] to an E S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.T he application of biocatalysts in the production of optically pure compounds has attracted much attention during the past few decades. Enantioselectivity (E) is one of the key parameters that define the usefulness of enzymes in the corresponding industrial synthesis of fine chemicals. Thus, identification of enzymes with high enantioselectivity for a desired transformation is important. In nature, biocatalysts with high enantioselectivity for specific industrial applications are not readily available. Directed evolution has become a routine approach for developing such novel biocatalysts, and a plethora of successful examples have been reported (1-6). A recent survey regarding the location of mutations that improve enzyme properties shows that to manipulate the enantioselectivity of enzymes, closer mutations are more effective than distant mutations (7). With the availability of structural information, the methodology of CASTing (combinatorial activesite saturation test) used in an iterative manner has been particularly successful in tailoring enzyme enantioselectivity (8-11).Microbial halohydrin dehalogenases attract a great deal of attention for their ability to produce optically pure epoxides and halohydrins (12, 13). 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 act...

Covalent immobilization of halohydrin dehalogenase for efficient synthesis of epichlorohydrin in an integrated bioreactor

“…Halohydrin dehalogenase (HHDH, EC 4.5.1.X) can catalyze the dehalogenation of vicinal halohydrins to produce their corresponding epoxides . HHDH‐mediated dehalogenation has several advantages over the other routes, such as eco‐friendly reaction conditions, a cheap starting material, and the lack of necessity for an externally added cofactor . In the past few decades, a number of HHDHs have been recognized as useful tools for the production of pharmaceutical intermediates and the biodegradation of toxic chlorinated organic compounds.…”

“…In the past few decades, a number of HHDHs have been recognized as useful tools for the production of pharmaceutical intermediates and the biodegradation of toxic chlorinated organic compounds. HHDH has been proven to be a valuable alternative to chemical catalysts now used to dehalogenate halohydrins to produce epoxides …”

Biodegradation of 1,2,3-Trichloropropane to Valuable (S)-2,3-DCP Using a One-Pot Reaction System

“…We apply the recombinant expression of both DhaA31 and the wild-type HheC enzymes in pBAD system as formerly reported by using Escherichia coli MC1061 as host cells [19]. Cells were harvested by centrifugation (5000 x g, 20 min) at 4 °C and were washed once with 10 mM phosphate buffer (pH 8.0).…”

Recent advances on halohydrin dehalogenases—from enzyme identification to novel biocatalytic applications

“…Each monomer’s active site is buried within the enzyme and is connected with the bulk solvent via a substrate entrance tunnel. This tunnel is differently shaped in the crystallized HHDHs and —at least for HheC— various studies demonstrate that residues of the entrance tunnel are involved in determining the enzymes’ activity and enantioselectivity (Schallmey et al 2013 ; Guo et al 2015 ; Wang et al 2015b ). The position of the catalytic residues Ser, Tyr, and Arg within the active sites is also highly conserved.…”