Computational protocol: Conformational diversity and enantioconvergence in potato epoxide hydrolase 1††Electronic supplementary information (ESI) available: Further details on calibration of our simulations, QM data and RMSD plots from the MD simulations, further experimental data, and all EVB parameters used to model styrene oxide hydrolysis in this study. See DOI: 10.1039/c6ob00060fClick here for additional data file.

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[…] The enzyme variant R-C1B1 was originally isolated from a laboratory evolution for StEH1 variants displaying enrichment of the (R)-enantiomer of the diol product from the enzyme-catalysed hydrolysis of racemic (2,3-epoxypropyl)benzene (EPB). This variant has accumulated four active-site mutations, specifically W106L, L109Y, V141K and I155V. In addition to the altered regioselectivity in the hydrolysis of EPB, this variant also exhibits a change in the regioselectivity during hydrolysis of (R)-SO and was therefore included in this study.Wild-type StEH1 and the R-C1B1 variant were expressed in Escherichia coli XL1-Blue (Stratagene Corp.), and purified by Ni(II)-IMAC and size exclusion chromatography as described previously. Protein concentrations were determined through measuring UV absorption at 280 nm, using an extinction coefficient (ε) based on the value for wild-type StEH1, corrected by:1εvariant = εWT + (Trp × 5500)+(Tyr × 1490)+(Cys × 125) The R-C1B1 variant of StEH1 was crystallized by hanging drop vapour diffusion against a 1 ml reservoir at 20 °C. The 3 μl drop was prepared by mixing 2 μl protein solution (5 mg ml–1 in 30 mM Tris-HCl, pH 7.4) with 1 μl reservoir solution containing 18% (w/v) PEG 5000-monomethyl ether, 0.1 M Tris-HCL, pH 8.0, and 5% (v/v) dioxane.Crystals of R-C1B1 were flash-frozen in liquid nitrogen without additional cryo-protection. Crystallographic data were collected at 100 K at beamline I04 of the Diamond Light Source (Didcot, UK). Data were indexed and integrated on site with XDS– and scaled with SCALA from the CCP4 suite of programs., The crystals belong to space group P212121 and contain two identical polypeptide chains per asymmetric unit. Data collection statistics are given in .The structure of the R-C1B1 variant was solved by molecular replacement with PHASER and wild-type StEH1 as a search model (PDB ID: ; 2CJP). Manual model building was performed with COOT and alternated with restrained refinement in REFMAC5. A set of ∼5% randomly selected reflections were used for monitoring R free. Water molecules were added in COOT.The final model contains residues 2–321 for both the A and B chain, respectively, and 642 water molecules. A dioxane molecule from the crystallization solution is bound to the active site of both R-C1B1 molecules present in the asymmetric unit. The model has good stereochemistry, with >98% of the residues found in the most favourable and 0.3% in the disallowed region of the Ramachandran plot, respectively. The refinement statistics are given in . The crystallographic data and structure of the StEH1 R-C1B1 variant have been deposited in the Protein Data Bank with the accession code ; 4UFN. This crystal structure provided the starting point for all subsequent simulations of the activity of the R-C1B1 variant presented in this work, following exactly the same procedures as used for wild-type StEH1. […]

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Conformational diversity and enantioconvergence in potato epoxide hydrolase 1††Electronic supplementary information (ESI) available: Further details on calibration of our simulations, QM data and RMSD plots from the MD simulations, further experimental data, and all EVB parameters used to model styrene oxide hydrolysis in this study. See DOI: 10.1039/c6ob00060fClick here for additional data file.

2016 Organic & Biomolecular Chemistry
PMCID: 5315018
PMID: 27049844
DOI: 10.1039/c6ob00060f

Pipeline specifications

Software tools XDS, CCP4, Coot, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Solanum tuberosum
Chemicals Amino Acids, Carbon