Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity

Abstract

HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G.

Biocatalytically active and stable cross-linked enzyme crystals of halohydrin dehalogenase HheG by protein engineering

“…To evaluate enantioselectivities, product enantiomeric excesses were obtained from 4 h samples by chiral GC described elsewhere. [17] To assess activities in different organic co-solvents, reactions contained 25% (v/v) of co-solvents such as ethanol, methanol, 2-propanol, acetonitrile, dimethylsulfoxide, dimethylformamide. Sampling of duplicate reactions was performed after 4 and 24 h and analyzed by achiral GC.…”

“…[16] A major drawback for application, however, is the enzyme's low stability (apparant melting temperature Tm of only 38 °C) and its low organic solvent tolerance. [17] Based on protein engineering of HheG, variants with amino acid exchanges at position T123 have been obtained displaying up to 14 K higher Tm as well as up to three-fold higher activity, resulting also in a slight increase in organic solvent resistance. [17] To complement our protein engineering efforts with HheG, we herein report an orthogonal strategy to the stabilization of HheG using immobilization.…”

“…[17] Based on protein engineering of HheG, variants with amino acid exchanges at position T123 have been obtained displaying up to 14 K higher Tm as well as up to three-fold higher activity, resulting also in a slight increase in organic solvent resistance. [17] To complement our protein engineering efforts with HheG, we herein report an orthogonal strategy to the stabilization of HheG using immobilization. [18] Common enzyme immobilization methods include carrier binding, polymer entrapment, and carrier-free immobilization methods based on cross-linking such as cross-linked enzyme crystals (CLECs) or cross-linked enzyme aggregates (CLEAs).…”

Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2

“…It is worth mentioning the new hydrogen bond interaction between residues P170 and T72 identified with the Arpeggio program [28]. The increase in thermal stability in energy minima B can be explained by a partial cover of the active site by residues 170-186, which increases the buried surface area of the enzyme [29,30]. Despite the huge rearrangement of helices αE and αF in minima B, the residues that compose the halidebinding site and the catalytic triad maintained similar positions than those observed in the X-ray structure, making possible the reaction to occur at 57°C (Fig.…”

The Depth-Dependent Mechanical Behavior of Anisotropic Native and Cross-Linked HheG Enzyme Crystals

“…Conversion of the cyclohexene oxide using HheG CLECs was carried out at a 1 mL scale, and the reactions were analyzed by gas chromatography (GC) [37]. Reactions were performed at 22 • C and 900 rpm in a Tris•SO 4 buffer (50 mM, pH 7.0) containing cyclohexene oxide (20 mM) and sodium azide (40 mM) as a nucleophile.…”