6A6T
Crystal structure of the modified fructosyl peptide oxidase from Aspergillus nidulans with R61G mutation
Summary for 6A6T
| Entry DOI | 10.2210/pdb6a6t/pdb |
| Descriptor | Fructosyl amine: oxygen oxidoreductase, FLAVIN-ADENINE DINUCLEOTIDE, SULFATE ION, ... (5 entities in total) |
| Functional Keywords | fructosyl peptide, oxidoreductase, aspergillus nidulans, fad-binding protein |
| Biological source | Aspergillus nidulans |
| Total number of polymer chains | 1 |
| Total formula weight | 50552.42 |
| Authors | Ogawa, N.,Maruyama, Y.,Itoh, T.,Hashimoto, W.,Murata, K. (deposition date: 2018-06-29, release date: 2019-05-15, Last modification date: 2024-10-30) |
| Primary citation | Ogawa, N.,Kimura, T.,Umehara, F.,Katayama, Y.,Nagai, G.,Suzuki, K.,Aisaka, K.,Maruyama, Y.,Itoh, T.,Hashimoto, W.,Murata, K.,Ichimura, M. Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis. Sci Rep, 9:942-942, 2019 Cited by PubMed 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. PubMed: 30700768DOI: 10.1038/s41598-018-37806-x PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.901 Å) |
Structure validation
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