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.

Manetti, F., Mileto, D., Corelli, F., Soro, S., Palocci, C., Cernia, E., et al. (2000). Design and Realization of a Tailor-made Enzyme to Modify the Molecular Rrecognition of 2-Arylpropionic Esters by Candida rugosa Lipase. BIOCHIMICA ET BIOPHYSICA ACTA, 1543(1), 146-158 [10.1016/S0167-4838(00)00185-0].

Design and Realization of a Tailor-made Enzyme to Modify the Molecular Rrecognition of 2-Arylpropionic Esters by Candida rugosa Lipase

Manetti, Fabrizio;Corelli, Federico;Botta, Maurizio
2000-01-01

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.
2000
Manetti, F., Mileto, D., Corelli, F., Soro, S., Palocci, C., Cernia, E., et al. (2000). Design and Realization of a Tailor-made Enzyme to Modify the Molecular Rrecognition of 2-Arylpropionic Esters by Candida rugosa Lipase. BIOCHIMICA ET BIOPHYSICA ACTA, 1543(1), 146-158 [10.1016/S0167-4838(00)00185-0].
File in questo prodotto:
File Dimensione Formato  
2000BiochimBiophysActaTailorMade.pdf

non disponibili

Tipologia: Post-print
Licenza: NON PUBBLICO - Accesso privato/ristretto
Dimensione 651.6 kB
Formato Adobe PDF
651.6 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/21525
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo