Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi

Abstract

Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N(alpha)-fructosyl valyl-histidine (alpha-keto-amine) and N(epsilon)-fructosyl lysine (epsilon-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both alpha-keto-amine and epsilon-keto-amine, and (2) enzymes that preferably oxidize alpha-keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric ( M(r)=50,000), was most active at 40 degrees C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K(m) values for N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine were 2.30 and 1.69 mM, respectively. N(alpha)-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A(1C), the N-terminal valine residue of the beta-subunit of which is glycated.

Enzymes used for the determination of HbA1C

“…Fru-hexapeptide, Fru-ValHis and valyl-histidine (ValHis) were measured by HPLC on an Amide-80 column (£ 4.6 mm  25 cm; Tosoh, Japan) with 30% KH 2 PO 4 (10 mM) in acetonitrile as a solvent at a flow rate of 1.0 ml/min with monitoring of the absorbance at 205 nm [14]. Fru-hexapeptide, Fru-ValHis and ValHis were used as controls for the HPLC analysis.…”

“…showed specificity toward N e -(1-deoxyfructosyl)-N a -Z-Lys, N -(1-deoxyfructosyl)-Val (Fru-Val) and/or fructosyl propylamine [12,9,13]. Recently, we screened a fungal culture collection for fructosyl peptide oxidase (FPOX) using N -(1-deoxyfructosyl)-Val-His (Fru-ValHis) as a substrate [14]. We found that the cell extracts of several genera of fungi showed FPOX activity and then purified and characterized two FPOXs; one from Eupenicillium terrenum ATCC18547 and one from Coniochaeta sp.…”

An enzymatic method for the determination of hemoglobinA1C

“…Recently, we screened a fungal culture collection and isolated a novel type of FAOX from Eupenicillium terrenum ATCC18547 and from Coniochaeta sp. NISL9330 (Hirokawa et al 2003a). These deglycating enzymes showed specificity towards both fructosyl amino acids and fructosyl peptides such as N-(1-deoxyfructosyl)-Val-His (Fru-ValHis), while previously isolated FAOXs were only active against fructosyl amino acids (Hirokawa et al 2003b).…”

X-ray structures of fructosyl peptide oxidases revealing residues responsible for gating oxygen access in the oxidative half reaction

“…Fructosyl amine oxidases (FAOXs) are FAD-containing enzymes that catalyze the oxidative deglycosylation of fructosyl amino acids such as fructosyl valine and/or ε-fructosyl lysine, which are the degradation products of HbA1c and GA, respectively. As members of the FAOX family, fructosyl peptide oxidases (FPOXs) are also known as candidates for diagnostic enzymes 6 . A few promising FPOXs derived from Eupenicillium terrenum (EtFPOX) and Coniochaeta sp.…”