6533b7ddfe1ef96bd1273d7e
RESEARCH PRODUCT
Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel
Alfonso GautieriFederica RigoldiAnna CarboneAnna CarboneEmilio ParisiniAlberto RedaelliTiziano BandieraArchimede TorrettaStefano Doninisubject
access tunnel biosensor diabetes fructosyl peptide oxidase rational enzyme designBioengineeringPeptidebiosensorApplied Microbiology and Biotechnologychemistry.chemical_compoundCatalytic DomainEnzyme Stabilityfructosyl peptide oxidasechemistry.chemical_classificationdiabetesbiologyPoint mutationRational designProteolytic enzymesAlbuminActive siteSettore CHIM/08 - Chimica FarmaceuticaEnzymeBiochemistrychemistryrational enzyme designbiology.proteinAmino Acid OxidoreductasesGlycated hemoglobinaccess tunnelBiotechnologydescription
Fructosyl peptide oxidases (FPOXs) are enzymes currently used in enzymatic assays to measure the concentration of glycated hemoglobin and albumin in blood samples, which serve as biomarkers of diabetes. However, since FPOX are unable to work directly on glycated proteins, current enzymatic assays are based on a preliminary proteolytic digestion of the target proteins. Herein, to improve the speed and costs of the enzymatic assays for diabetes testing, we applied a rational design approach to engineer a novel enzyme with a wider access tunnel to the catalytic site, using a combination of Rosetta design and molecular dynamics simulations. Our final design, L3_35A, shows a significantly wider and shorter access tunnel, resulting from the deletion of five-amino acids lining the gate structures and from a total of 35 point mutations relative to the wild-type (WT) enzyme. Indeed, upon experimental testing, our engineered enzyme shows good structural stability and maintains significant activity relative to the WT.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2020-01-01 |