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

Abstract

Halohydrin dehalogenases (HHDHs) are promising enzymes for application in biocatalysis due to their promiscuous epoxide ring-opening activity with various anionic nucleophiles. So far, seven different HHDH subtypes A to G have been reported with subtype D containing the by far largest number of enzymes. Moreover, several characterized members of subtype D have been reported to display outstanding characteristics such as high catalytic activity, broad substrate spectra or remarkable thermal stability. Yet, no structure of a D-type HHDH has been reported to date that could be used to investigate and understand those features on a molecular level. We therefore solved the crystal structure of HheD2 from gamma proteobacterium HTCC2207 at 1.6Å resolution and used it as a starting point for targeted mutagenesis in combination with molecular dynamics (MD) simulation, in order to study the low thermal stability of HheD2 in comparison with other members of subtype D. This revealed a hydrogen bond between conserved residues Q160 and D198 to be connected with a high catalytic activity of this enzyme. Moreover, a flexible surface region containing two α-helices was identified to impact thermal stability of HheD2. Exchange of this surface region by residues of HheD3 yielded a variant with 10°C higher melting temperature and reaction temperature optimum. Overall, our results provide important insights into the structure-function relationship of HheD2 and presumably for other D-type HHDHs.

Characterization of a new G-type halohydrin dehalogenase with enhanced catalytic activity

“…A remarkable difference, however, can be observed in the three dimensional arrangement of alpha helices α6 and α7 in HheG-682 (residues 189-212) and HheG (residues 200-223) (Figure 7B). As previously reported [3,12] , residue F203 of HheG, which delimits the enzyme active site of HheG together with residues of the catalytic triad, is found in helix α6. Moreover, HheG-682 is missing the flexible loop (spanning residues 39-47 in HheG) that has been reported for HheG previously.…”

Biocatalytically Active and Stable Cross‐Linked Enzyme Crystals of Halohydrin Dehalogenase HheG by Protein Engineering

“…[11,12] Additionally, HheG displays αregioselectivity in the ring-opening of different styrene oxide derivates, [13] whereas other HHDHs exclusively attack the βcarbon of the epoxide ring. Solving the crystal structure of HheG [11] revealed a much broader active site for this enzyme compared to other HHDHs, [14][15][16][17] which explains the acceptance of bulkier substrates. This feature makes HheG very attractive for industrial application, e. g. for the synthesis of pharmaceutical building blocks.…”

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

“…[9,10] Additionally, HheG displays α-regioselectivity in the ring-opening of different styrene oxide derivates, [11] whereas other HHDHs exclusively attack the β-carbon of the epoxide ring. Solving the crystal structure of HheG [9] revealed a much broader active site for this enzyme compared to other HHDHs, [12][13][14][15] which explains the acceptance of bulkier substrates. This feature makes HheG very attractive for industrial application, e.g.…”