title: Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase.
abstract: Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different 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 structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.
authors.affiliation: Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via Aldo Moro snc, I-53100 Siena, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Manetti
authors.forename: D
authors.initials: D
authors.lastname: Mileto
authors.forename: F
authors.initials: F
authors.lastname: Corelli
authors.forename: S
authors.initials: S
authors.lastname: Soro
authors.forename: C
authors.initials: C
authors.lastname: Palocci
authors.forename: E
authors.initials: E
authors.lastname: Cernia
authors.forename: I
authors.initials: I
authors.lastname: D'Acquarica
authors.forename: M
authors.initials: M
authors.lastname: Lotti
authors.forename: L
authors.initials: L
authors.lastname: Alberghina
authors.forename: M
authors.initials: M
authors.lastname: Botta
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D004952:Esters / Q000378:metabolism; D007660:Ketoprofen / Q000737:chemistry; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009288:Naproxen / Q000737:chemistry; D011422:Propionates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004952:Esters; D011422:Propionates; D011994:Recombinant Proteins; D009288:Naproxen; D007660:Ketoprofen; D008049:Lipase