Fig 1.
Three-dimensional model of HvADH2 active site in complex with different docked ligands.
A) Complex with (S)-1-PheOH. B) LigPlot analysis of the interactions between docked (S)-1-PheOH and HvADH2 model. The substrate is in purple. Hydrophobic contacts are shown as dark red arches. C) Complex with (S)-flurbiprofenol. D) Complex with (S)-flurbiprofenol, surface representation. The ligands are represented as white sticks or spheres. The NAD+ cofactor is in yellow and Zn2+ is represented as a pink sphere. Important residues are shown in green; the αC of G294 is shown as a sphere. The π-π staking interactions are shown by dotted lines and H-bonds by dashed lines.
Fig 2.
Sequence alignment of HvADH2 with top 9 homologues.
3KRT: putative crotonyl CoA reductase from Streptomyces coelicolor. 1KOL: Formaldehyde dehydrogenase from Pseudomonas putida. 2DPH: Formaldehyde dismutase from Pseudomonas putida. 1HF3: liver alcohol dehydrogenase from Equus caballus. 1H2B alcohol dehydrogenase from Aeropyrum pernix. 4A10: 2-octenoyl-CoA carboxylase reductase from Streptomyces sp. JS360. 2CDB: glucose dehydrogenase from Sulfolobus solfataricus. 1F8F: benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus. 1MAO: glutathione-dependent formaldehyde dehydrogenase from Homo sapiens.
Fig 3.
F108L docked (F85 blue spheres and L108 purple spheres; NAD+, yellow sticks) with (S)-flurbiprofenol (white spheres), surface view.
The distance from the hydroxyl oxygen to the catalytic zinc (O-Zn) is 4.4 Å, and the distance from the substrate α-carbon to the C4 of the nicotinamide ring (αC-C4), is 5.9.
Table 1.
Activity of purified wild-type and F108 variants of HvADH2 with rac-flurbiprofenol.
Fig 4.
Substrate specificity of purified F108G variant compared to WT HvADH2.
Buffer conditions: 4 M KCl, 50 mM Gly-KOH, pH 10.0.
Fig 5.
Docking of (R)-1-PheOH (white spheres).
A) Catalytic site of the WT enzyme (F85 and F108 green spheres, NAD+, yellow sticks) shows stabilization of the substrate aromatic ring by F108. The distance from the hydroxyl oxygen to the catalytic zinc (O-Zn) is 4.6 Å, and the distance from the substrate α-carbon to the C4 of the nicotinamide ring (αC-C4), is 5.7 Å. B) Catalytic site of the F108G variant. The distance from the hydroxyl oxygen to the catalytic zinc (O-Zn) is 2.5 Å, and the distance from the substrate α-carbon to the C4 of the nicotinamide ring (αC-C4), is 4.0 Å.