Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis.
Docking models of fructosyl amine oxidase (FAOD) from the marine yeast Pichia N1-1 (N1-1 FAOD) with the substrates fructosyl valine (f-Val) and fructosyl-(epsilon)N-lysine (f-(epsilon)Lys) were produced using three-dimensional protein model as reported previously (Miura et al., 2006, Biotechnol. Lett., 28, 1895-1900). The residues involved in recognition of substrates were proposed, particularly Asn354, which interacts closely with f-(epsilon)Lys, but not with f-Val. Substitution of Asn354 to histidine and lysine simultaneously resulted in an increase in activity of f-val and a decrease in activity of f-(epsilon)Lys and thus, increasing the specificity for f-Val from 13- to 19-fold. In addition to creating two mutant FAODs with great potential for the measurement of glycated hemoglobin, we have provided the first structural model of substrate binding with eukaryotic FAOD, which is expected to contribute to further investigation of FAOD.
Miura
Seiji
S
Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi 185-8688, Japan.
Ferri
Stefano
S
Tsugawa
Wakako
W
Kim
Seungsu
S
Sode
Koji
K
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D002021:Buffers; D006442:Glycated Hemoglobin / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010843:Pichia / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050879:Sarcosine Oxidase / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014633:Valine / Q000031:analogs & derivatives* / Q000378:metabolism
D000594:Amino Acid Oxidoreductases; D001216:Asparagine; D002021:Buffers; D006442:Glycated Hemoglobin; D008239:Lysine; D011994:Recombinant Proteins; D050879:Sarcosine Oxidase; D014633:Valine; C045420:fructosylvaline; C033186:fructosyl-lysine; C103012:amadoriase