At the moment, there are approx. 100 published papers investigating halohydrin dehalogenases from different aspects; enzymology, molecular biology and reactions they can catalyse. Unquestionably, ...these enzymes are of great importance and hold an immense potential due to the wide spectrum of different compounds that can be synthesized by their action. These compounds, such as chiral epoxides, β‐substituted alcohols, oxazolidinones etc., significantly enrich the chemist's toolbox and, moreover, open the possibility for the synthesis of even more complex compounds. Still, there are many unknowns, and it is the purpose of this work to demonstrate the possibilities and bottlenecks, in scientific sense, that could further help in broadening the applicative potential of these fascinating enzymes.
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.
Although the application of organic solvents in biocatalysis is well explored, in‐depth understanding of the interactions of solvent with proteins, in particular oligomeric ones, is still scant. ...Understanding these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, the homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO/water solvent mixtures. DMSO, the most commonly used co‐solvent for biocatalytic transformations, was found to act as a mixed‐type inhibitor with a prevalent competitive contribution. Even 5 % (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser‐Tyr catalytic residues forming alternate H‐bonds with them. Stability measurements paired with differential scanning calorimetry, dynamic light scattering methods and MD studies revealed that HheC maintains its structural integrity with as much as 30 % (v/v) DMSO.
Pros and cons considered: Halohydrin dehalogenases (HHDHs) are versatile enzymes employed in the synthesis of chiral building blocks. To increase substrate solubility, DMSO stands out as the most commonly used co‐solvent. Here we found that DMSO acts as an inhibitor of the enzyme HheC. To understand the observed phenomena at the molecular level, in‐depth experimental studies were performed in combination with molecular dynamics simulations.
Invited for the cover of this issue are Zlatko Brkljača, Maja Majerić Elenkov and co‐workers at the Ruđer Bošković Institute and University of Zagreb. The image depicts the enzyme halohydrin ...dehalogenase HheC, which is made up of four identical subunits, with marked catalytic residues and volumetric maps of water and DMSO in the active site. Read the full text of the article at 10.1002/chem.202201923.
“The first comprehensive study of the mechanisms by which DMSO, the most commonly used co‐solvent in the halohydrin dehalogenase‐catalysed reactions, influences the activity and stability of the enzyme HheC.” This and more about the story behind the front cover can be found in the article at 10.1002/chem.202201923).
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
An efficient dynamic kinetic resolution protocol using a single enzyme is described. Both the kinetic resolution and substrate racemization are catalyzed by halohydrin dehalogenase from Agrobacterium ...radiobacter AD1 (HheC). The HheC‐catalyzed reaction of epibromohydrin and 2‐bromomethyl‐2‐methyloxirane with sodium cyanate afforded 5‐substituted 2‐oxazolidinones in high yields (97% and 87%) and high optical purity (89% and >99% ee) in the presence of catalytic amounts of bromide ion. These compounds are valuable building blocks with diverse synthetic applications.
Easy virtue: Halohydrin dehalogenase is a highly promiscuous enzyme that can catalyze enantioselective epoxide ring opening with at least nine different anionic nucleophiles (see scheme). Its ...capacity to form carbon–nitrogen, carbon–oxygen, carbon–sulfur, and carbon–carbon bonds makes it possible to use this enzyme for the preparation of a range of highly enantioenriched β‐substituted alcohols or derivatives thereof, including cyanoalcohols, nitroalcohols, and oxazolidinones.
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