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-N-alpha-valine (f-(alpha)Val), a model compound of hemoglobin A(1c) (HbA(1c)). Kinetic analysis of these mutant FAODs indicated that these His51 variants had decreased VmaxKm-1 values for fructosyl-N-epsilon-lysine (f-(epsilon)Lys) compared to the wild-type enzyme, while the VmaxKm-1 values for f-(alpha)Val 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-(alpha)Val over f-(epsilon)Lys and higher activity toward f-(alpha)Val than the single mutants and the wild-type. In order to develop a biosensor for the measurement of HbA(1c), an FAOD enzyme specific to f-(alpha)Val is required to avoid influence of f-(epsilon)Lys derived from other glycated proteins. Our results support the proposed 3D model, and the resulting f-(alpha)Val-specific Mutants are expected to be applied to the enzymatic measurement of HbA(1c). (C) 2008 Elsevier Inc. All rights reserved.