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.

Keywords: Diabetes; Eupenicillium terrenum; Fructosyl peptide oxidase (FPOX); Hemoglobin A1c (HbA1c); Protein engineering; Substrate specificity.