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Cumulative effect of amino acid substitution for the development of fructosyl valine-specific fructosyl amine oxidase

Paper ID Volume ID Publish Year Pages File Format Full-Text
17858 42701 2009 5 PDF Available
Title
Cumulative effect of amino acid substitution for the development of fructosyl valine-specific fructosyl amine oxidase
Abstract

Site-directed mutagenesis was carried out at the putative active site of fructosyl amine oxidase (FAOD) to improve its substrate specificity based on information from the structural model. Substitution of the His51 residue with other amino acids predicted to interact with the substrate resulted in mutant FAODs with improved specificity for fructosyl-αN-valine (f-αVal), a model compound of hemoglobin A1c (HbA1c). Kinetic analysis of these mutant FAODs indicated that these His51 variants had decreased VmaxKm−1VmaxKm−1 values for fructosyl-ɛN-lysine (f-ɛLys) compared to the wild-type enzyme, while the VmaxKm−1VmaxKm−1 values for f-αVal remained unaffected or were increased. Among the 19 variants at His51, His51Lys/Arg was combined with previously reported mutants, such as Asn354His [Miura S, Ferri S, Tsugawa W, Kim S, Sode K. Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Eng Des Sel 2008;21:233–9]; the His51/Asn354 double mutant showed a greater improvement in the specificity for f-αVal over f-ɛLys and higher activity toward f-αVal than the single mutants and the wild-type. In order to develop a biosensor for the measurement of HbA1c, an FAOD enzyme specific to f-αVal is required to avoid influence of f-ɛLys derived from other glycated proteins. Our results support the proposed 3D model, and the resulting f-αVal-specific mutants are expected to be applied to the enzymatic measurement of HbA1c.

Keywords
Biosensor; Diabetes; Fructosyl amine oxidase; Hemoglobin A1c (HbA1c); Substrate specificity
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Cumulative effect of amino acid substitution for the development of fructosyl valine-specific fructosyl amine oxidase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 44, Issue 1, 6 January 2009, Pages 52–56
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us