Biocatalytic transformations in organic synthesis often require the use of organic solvents to improve substrate solubility and promote the product formation. Halohydrin dehalogenases (HHDHs) are ...enzymes that catalyze the formation and conversion of epoxides, important synthetic class of compounds that are often sparingly soluble in water and prone to hydrolysis. In this study, the activity, stability, and enantioselectivity of HHDH from
Agrobacterium radiobacter
AD1 (HheC) in form of cell-free extract were evaluated in various aqueous-organic media. A correlation was discovered between the enzyme activity in the ring-closure reaction and logP of the solvent. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. The results revealed a high enzyme compatibility with hydrophobic solvents (e.g.,
n
-heptane) in terms of activity and stability. Regarding the HHDH applicability in an organic medium, inhibitions by a number of solvents (e.g., THF, toluene, chloroform) proved to be a more challenging problem than the protein stability, especially in the ring-opening reaction, thus suggesting which solvents should be avoided. In addition, solvent tolerance of the thermostable variant ISM-4 was also evaluated, revealing increased stability and to a lesser extent enantioselectivity compared to the wild-type. This is the first time such a systematic analysis has been reported, giving insight into the behavior of HHDHs in nonconventional media and opening new opportunities for the future biocatalytic applications.
Key points
•
HheC performs better in the presence of hydrophobic than hydrophilic solvents.
•
Enzyme activity in the PNSHH ring-closure reaction is a function of the logP.
•
Thermostability of ISM-4 variant is accompanied by superior solvent tolerance.
Graphical Abstract
Ten different fluorinated aromatic epoxides have been tested as potential substrates for halohydrin dehalogenase (HHDH) HheC. The majority of investigated epoxides are useful building blocks in ...synthetic chemistry applications, with a number of them being polysubstituted. Moderate to high enantioselectivities (
= 15 → 200) were observed in azidolysis, allowing the synthesis of enantioenriched (
)-azido alcohols containing fluorine in the molecule. In the case where a reaction runs over 50% conversion, enantiopure (
)-epoxides are also available. While
-F-styrene oxide was easily converted into a product, a sterically challenging
-CF
-derivative was not accepted by HheC.
probing of the binding site indicates that, in order to accommodate an
-CF
-derivative in the HheC active site, it is necessary to eliminate steric hindrance. Hence, we extended our research by probing several available HheC variants containing relevant modifications in the active site. The active mutant P84V/F86P/T134A/N176A (named HheC-M4) was identified, showing not only high activity towards
-CF
-styrene oxide, but also inverted enantioselectivity (
= 27). Since (
)-enantioselective HHDHs are rare and therefore valuable for their synthetic application, this enzyme was screened on the initial panel of substrates. The observed (
)-enantioselectivity (
= 1-111) is ascribed to the formation of the additional space by introduced mutations in HheC-M4, which is also confirmed by classical MD simulations. Successive molecular docking demonstrated that this newly formed tunnel located close to the protein surface is a critical feature of HheC-M4, representing a novel binding site.
