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PDBsum entry 6a6t
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Oxidoreductase
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PDB id
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6a6t
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References listed in PDB file
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Key reference
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Title
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Creation of haemoglobin a1c direct oxidase from fructosyl peptide oxidase by combined structure-Based site specific mutagenesis and random mutagenesis.
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Authors
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N.Ogawa,
T.Kimura,
F.Umehara,
Y.Katayama,
G.Nagai,
K.Suzuki,
K.Aisaka,
Y.Maruyama,
T.Itoh,
W.Hashimoto,
K.Murata,
M.Ichimura.
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Ref.
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Sci Rep, 2019,
9,
942.
[DOI no: ]
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PubMed id
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Abstract
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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.
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