Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis

Abstract

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.

Molecular Dynamics Simulations Provide Insights into Structure and Function of Amadoriase Enzymes

“…Finally, we observe that both f-lys and f-val are highly unstable in the N1-1-FAOD enzyme, as suggested by the low population of the major conformational cluster (46 % for f-lys and 18 % for f-val) and by the high mobility of the substrates in the binding pocket, also involving the otherwise usually stable sugar moiety. This result is in accordance with the experimental findings [4,6], which show that N1-1-FAOD has the worst K m and V max when compared with all the FAOX considered in this study (see also Table 3). …”

“…Structure-based sequence alignment of the five selected FAOD enzymes. Amadoriase I and Amadoriase II sequences from Aspergillus Fumigatus [30][31][32], PnFPOX from Phaeospheria nodorum [33], and N1-1-FAOD from Pichia species N11 [4,7] were aligned using ClustalW 1.82 [34][35][36] and colored using ESPript 3.0 [37]. White in a red box shows strict identity, while red in a white box indicate similarity.…”

“…In earlier works [4][5][6], the Sode group has addressed this issue by performing extensive mutagenesis experiments on the N1-1-FAOD from Pichia marine yeast, especially at those sites that are putatively involved in ligand binding (e.g., His51 and Asn374). As a result, they managed to provide the enzyme with improved selectivity for fructosyl valine over fructosyl lysine, a favorable feature for the detection of HbA1c.…”

Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase II)

“…Many groups have reported efforts, with the help of computerized models, to improve substrate selectivity of FAOX enzymes for fructosylvaline to develop enzymatic assays utilizing proteolytic digests of glycated hemoglobin (38). The FAOX-II structure will aid in this endeavor.…”

Biomolecular Engineering of Biosensing Molecules —The Challenges in Creating Sensing Molecules for Glycated Protein Biosensing—

“…By creating a structural model and carrying out docking studies, we identified the amino acid residue Asn354 as interacting with the potential substrates. 33,34 Substitution of Asn354 to histidine or lysine simultaneously increased the enzyme's activity towards f-A Val and decreased that towards f-X Lys, thus greatly improving its specificity for f-A Val. Similarly, substitution of the His51 residue also produced mutants with significantly improved specificity for f-A Val.…”