Fructosyl peptide oxidases (FPOXs) are enzymes currently used in enzymatic assaysto measure the concentration of glycated hemoglobin and albumin in blood samples,which serve as biomarkers of diabetes. However, since FPOX are unable to workdirectly on glycated proteins, current enzymatic assays are based on a preliminaryproteolytic digestion of the target proteins. Herein, to improve the speed and costsof the enzymatic assays for diabetes testing, we applied a rational design approachto engineer a novel enzyme with a wider access tunnel to the catalytic site, using acombination of Rosetta design and molecular dynamics simulations. Our final design,L3_35A, shows a significantly wider and shorter access tunnel, resulting from thedeletion of fiveamino acids lining the gate structures and from a total of 35 pointmutations relative to the wildtype (WT) enzyme. Indeed, upon experimental testing,our engineered enzyme shows good structural stability and maintains significantactivity relative to the WT

F. Rigoldi, S. Donini, A. Torretta, A. Carbone, A. Redaelli, T. Bandiera, et al. (2020). Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel. BIOTECHNOLOGY AND BIOENGINEERING.

Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel

A. Carbone;
2020

Abstract

Fructosyl peptide oxidases (FPOXs) are enzymes currently used in enzymatic assaysto measure the concentration of glycated hemoglobin and albumin in blood samples,which serve as biomarkers of diabetes. However, since FPOX are unable to workdirectly on glycated proteins, current enzymatic assays are based on a preliminaryproteolytic digestion of the target proteins. Herein, to improve the speed and costsof the enzymatic assays for diabetes testing, we applied a rational design approachto engineer a novel enzyme with a wider access tunnel to the catalytic site, using acombination of Rosetta design and molecular dynamics simulations. Our final design,L3_35A, shows a significantly wider and shorter access tunnel, resulting from thedeletion of fiveamino acids lining the gate structures and from a total of 35 pointmutations relative to the wildtype (WT) enzyme. Indeed, upon experimental testing,our engineered enzyme shows good structural stability and maintains significantactivity relative to the WT
Settore CHIM/08 - Chimica Farmaceutica
F. Rigoldi, S. Donini, A. Torretta, A. Carbone, A. Redaelli, T. Bandiera, et al. (2020). Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel. BIOTECHNOLOGY AND BIOENGINEERING.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10447/432314
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