Atsushi Ichiyanagi

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.

Synthesis and inhibitory activity of substrate-analog fructosyl peptide oxidase inhibitors

Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized. These inhibitors mimic N(α)-fructosyl-L-valine (Fru-Val), [N(α)-fructosyl-L-valyl]-L-histidine (Fru-ValHis), and N(ε)-fructosyl-L-lysine (εFru-Lys), respectively. The secondary nitrogen atom in the natural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a-c with a sulfur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a-c act as competitive inhibitors against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782 μM were obtained for 1a-c, respectively.

Crystallization and preliminary crystallographic analysis of two eukaryotic fructosyl peptide oxidases

Fructosyl peptide oxidase (FPOX) catalyses the oxidation of α-glycated dipeptides such as N(α)-(1-deoxy-D-fructos-1-yl)-L-valyl-L-histidine (Fru-ValHis) and is used in the diagnosis of diabetes mellitus. Here, two thermostable mutants of FPOX, CFP-T7 and EFP-T5M, were crystallized by the sitting-drop vapour-diffusion method. The crystal of CFP-T7 belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 110.09, c = 220.48 Å, and that of EFP-T5M belonged to the monoclinic space group P2(1), with unit-cell parameters a = 43.00, b = 230.05, c = 47.27 Å, β = 116.99°. The crystals of CFP-T7 and EFP-T5M diffracted to 1.8 and 1.6 Å resolution, respectively.