Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase

Within a research project aimed at probing the substrate specificity and th e enantioselectivity of Candida rugosa lipase (CRL), computer modeling stud ies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)pro pionate (Ketoprofen methyl ester) have been carried out in order to identif y which amino acids are essential to the enzyme/substrate interaction. Diff erent binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The s tructural models of the computer generated complexes between CRL and the su bstrates enabled us to propose that Phe344 and Phe345, in addition to the r esidues constituting the catalytic triad and the oxyanion hole, are the ami no acids mainly involved in the enzyme-ligand interactions. To test the imp ortance of these residues for the enzymatic activity, site-directed mutagen esis of the selected amino acids has been performed, and the mutated enzyme s have been evaluated for their conversion and selectivity capabilities tow ard different substrates. The experimental results obtained in these biotra nsformation reactions indicate that Phe344 and especially Phe345 influence: CRL activity, supporting the findings of our theoretical simulations. (C) 2000 Elsevier Science B.V. All rights reserved.