Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design.

Jennifer Cassidy,Larah Bruen,Francesca Paradisi,Elena Rosini,Loredano Pollegioni,Gianluca Molla,Ivano Eberini

doi:10.1371/journal.pone.0187482

Abstract

An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

Highlights

Enzymes are appealing as a ‘green’ adjunct to chemical synthesis of pharmaceutical building blocks because of their broad specificity, enantioselectivity and ability to work under process conditions [1]
The quality of the ADH2 from Haloferax volcanii (HvADH2) homology model was assessed using the software ERRAT [23]: 30.1% of the protein structure model could be rejected at a 95% confidence level, but these regions are located on the protein surface and do not affect the overall conformation and substrate binding at the active site (S1 Fig)
The NAD+ cofactor and the conserved catalytic Zn2+ ion were modelled into the HvADH2 model based on their position and conformation observed in the structure of the formaldehyde dehydrogenase from Pseudomonas putida (PDB: 1kol, 26% sequence identity with HvADH2) [24]

Summary

Introduction

Enzymes are appealing as a ‘green’ adjunct to chemical synthesis of pharmaceutical building blocks because of their broad specificity, enantioselectivity and ability to work under process conditions [1]. Docking studies with the secondary aromatic alcohol, (S)-1-phenylethanol, ((S)-1-PheOH) allowed the identification of the residues involved in the substrate binding at the active site of HvADH2.

Results

Conclusion