Engineering an efficient mutant of Eupenicillium terrenum fructosyl peptide oxidase for the specific determination of hemoglobin A1c

Abstract

Fructosyl peptide oxidase (FPOX, EC 1.5.3) belongs to the family of oxidoreductases, which is used as a diagnostic enzyme for diabetes mellitus. FPOX has activities toward Fru-ValHis and Fru-Lys as model compounds for hemoglobin A1c (HbA1c) and glycated albumin, respectively. However, when the concentration of HbA1c is measured, the activity toward Fru-Lys will cause interference. In this study, we focused on the substrate specificity engineering of FPOX from Eupenicillium terrenum through computational and experimental methods with characteristics more suitable for HbA1c measurement in the blood. Based on structural knowledge of E. terrenum FPOX (PDB ID 4RSL) and molecular modeling results, residues His-377, Arg-62, Lys-380, and Tyr-261 were selected as mutagenesis sites. The best mutant with lower binding energy, stronger hydrophobic interactions, and more hydrogen bonds with Fru-ValHis and higher binding energy toward Fru-Lys was selected for experimental studies. To investigate the conformational changes in FPOX due to the mutation, molecular dynamics simulation was also performed. The genes encoding of native and engineered variants were cloned into pET-22b(+) and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant enzymes were purified and their kinetic properties were studied. Substitution of Tyr261 with Trp resulted in a mutant enzyme with improved specificity for Fru-ValHis, a model compound of HbA1c. The specific activity of mutant FPOX increased by 5.1-fold to 145.2 ± 3.2 U/mg for Fru-ValHis and decreased by 13.7-fold to 1.3 U/mg ± 0.9 for Fru-Lys compared to the native variant. Kinetics analysis indicated that Tyr261Trp FPOX mutant had 11.7-fold increase in K cat /K m for Fru-ValHis compared to the wild-type enzyme, while the K cat /K m for Fru-Lys diminished by 22.4-fold. In summary, our computational and experimental results suggested that the engineered FPOX is a good candidate to efficient determination of HbA1c.

Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel

“…Then, the FPOX enzyme binds and hydrolyzes the released fVH dipeptide, thus producing hydrogen peroxide which, in turn, is measured in a colorimetric assay using horseradish peroxidase and a suitable chromophore (Collard et al, 2008). Via a similar mechanism, FPOX enzymes can be used for the detection of glycated albumin, a short‐to mid‐term glycemic marker for diabetes (glycated albumin has a half‐life of 3 weeks; Shahbazmohammadi, Sardari, Lari, & Omidinia, 2019). To address the rapidly growing demand for more convenient monitoring of diabetes mellitus, efforts are aimed at developing enzymatic assays for glycated hemoglobin and albumin that do not require the preliminary proteolytic digestion of the target proteins.…”

Enzyme engineering and its industrial applications

“…The development of diagnostic enzymes is a growing field in pharmaceutical and medical studies. Shahbazmohammadi et al (63) used an engineered fructosyl peptide oxidase (FPOX) as a diagnostic enzyme for diabetes mellitus. Fructosylvalylhistidine, released from hemoglobin A1c, is a good biomarker for the diagnosis of diabetes mellitus and analysis of glycemic levels.…”

“…Several variants of FPOX were engineered to improve specificity for fructosylvalylhistidine as the substrate. The authors used both computational and experimental approaches to induce the mutation Tyr261Trp in FPOX, thus reducing interference of other amino acids by increasing its specificity (63).…”

Direct glucosone-based synthesis and HILIC-ESI-MS/MS characterization of N-terminal fructosylated valine and valylhistidine for validation of enzymatic HbA1c assays in the diagnosis of diabetes mellitus

“…However, increasing requirements on specificity, precision, throughput, and cost-effectiveness have led to the development of several new assay methods for monitoring HbA 1c . Some of these methods are based on the enzymatic quantification of HbA 1c [6][7][8][9]. The enzymatic methods are mainly facilitated by proteolytic digestion of hemoglobin and subsequent specific cleavage of fructosamines by fructosyl-amino acid oxidases or fructosyl-peptide oxidases generating hydrogen peroxide which can be measured photometrically by state-of-the-art procedures, mainly based on the oxidation of a leuco dye [6][7][8].…”

“…Some of these methods are based on the enzymatic quantification of HbA 1c [6][7][8][9]. The enzymatic methods are mainly facilitated by proteolytic digestion of hemoglobin and subsequent specific cleavage of fructosamines by fructosyl-amino acid oxidases or fructosyl-peptide oxidases generating hydrogen peroxide which can be measured photometrically by state-of-the-art procedures, mainly based on the oxidation of a leuco dye [6][7][8]. The substrates of these oxidases are fructosylated L-valine (Fru-Val) or fructosylated Lvalyl-L-histidine (Fru-Val-His), from which primarily the fructosylated dipeptide is used in commercially available enzymatic assays [10][11][12].…”