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Abstract
The currently available haemoglobin A1c (HbA1c) enzymatic assay consists of two specific steps: proteolysis of HbA1c and oxidation of the liberated fructosyl peptide by fructosyl peptide oxidase (FPOX). To develop a more convenient and high throughput assay, we devised novel protease-free assay system employing modified FPOX with HbA1c oxidation activity, namely HbA1c direct oxidase (HbA1cOX). AnFPOX-15, a modified FPOX from Aspergillus nidulans, was selected for conversion to HbA1cOX. As deduced from the crystal structure of AnFPOX-15, R61 was expected to obstruct the entrance of bulky substrates. An R61G mutant was thus constructed to open the gate at the active site. The prepared mutant exhibited significant reactivity for fructosyl hexapeptide (F-6P, N-terminal amino acids of HbA1c), and its crystal structure revealed a wider gate observed for AnFPOX-15. To improve the reactivity for F-6P, several mutagenesis approaches were performed. The ultimately generated AnFPOX-47 exhibited the highest F-6P reactivity and possessed HbA1c oxidation activity. HbA1c levels in blood samples as measured using the direct assay system using AnFPOX-47 were highly correlated with the levels measured using the conventional HPLC method. In this study, FPOX was successfully converted to HbA1cOX, which could represent a novel in vitro diagnostic modality for diabetes mellitus.
Highlights
Haemoglobin A1c (HbA1c) is a species of glycated haemoglobin (Hb) that is generated by non-enzymatic condensation between the amino group of the N-terminal valine of the Hb β-chain and glucose in the red blood cells[1]
The crystal structure of AnFPOX-15 illustrated that R61 apparently obstructs the entrance of bulky substrates such as F-6P into the active site
The generated R61G mutant acquired significant oxidation activity for F-6P, and the wider space of substrate gate in R61G mutant was confirmed via its crystal structure
Summary
Haemoglobin A1c (HbA1c) is a species of glycated haemoglobin (Hb) that is generated by non-enzymatic condensation between the amino group of the N-terminal valine of the Hb β-chain and glucose in the red blood cells[1] This in vitro diagnostic modality can serve as a biomarker for diabetes mellitus. Some of these enzymes, which exhibit reactivity for fructosyl dipeptides as F-VH are termed fructosyl peptide oxidase (FPOX) Because of their reactivity for F-VH, current HbA1c enzymatic assays often employ FPOX for a more specific measurement of HbA1c6. To meet the rapidly growing demand for more convenient monitoring of diabetes mellitus, we devised to develop protease-free enzymatic HbA1c in vitro diagnostics, namely “HbA1c direct enzymatic assay” This new assay is expected to bring several benefits. To construct such a novel system, we took the strategy to modify FPOX with direct oxidation activity for a whole HbA1c protein
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