Dataset Information

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis.

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.

SUBMITTER: Ogawa N

PROVIDER: S-EPMC6353924 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis.

Ogawa Noriyuki N Kimura Takehide T Umehara Fumi F Katayama Yuki Y Nagai Go G Suzuki Keiko K Aisaka Kazuo K Maruyama Yukie Y Itoh Takafumi T Hashimoto Wataru W Murata Kousaku K Ichimura Michio M

Scientific reports 20190130 1

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 deduce ...[more]

PMID: 30700768

Similar Datasets

Project description:ObjectivesWe investigated the influence of the preoperative haemoglobin A1c (HbA1c) value on the prognosis and pathology of patients with lung adenocarcinoma who underwent surgery.MethodsWe reviewed the medical records of 400 lung adenocarcinoma patients who underwent lobectomy with mediastinal lymph node dissection between 2009 and 2013 using a prospectively maintained database. We stratified 400 patients into 4 groups according to the preoperative HbA1c value as follows: HbA1c ≤ 5.9 (n = 296), 6.0 ≤ HbA1c ≤ 6.9 (n = 70), 7.0 ≤ HbA1c ≤ 7.9 (n = 21) and HbA1c ≥ 8.0 (n = 12). We compared the recurrence-free survival and overall survival (OS) among these 4 groups. Univariate and multivariate analyses were performed to identify the risk factors for recurrence.ResultsThe median follow-up period was 61.2 months. On comparing the recurrence-free survival and OS rates among these 4 groups, we found that these rates among patients in the HbA1c ≥ 8.0 group were significantly poorer compared with the other 3 groups (5-year recurrence-free survival: HbA1c ≤ 5.9, 70.4%; 6.0 ≤ HbA1c ≤ 6.9, 69.7%; 7.0 ≤ HbA1c ≤ 7.9, 70.7%; ≥8.0 HbA1c, 18.8%; P = 0.002; and 5-year OS: HbA1c ≤ 5.9, 88.7%; 6.0 ≤ HbA1c ≤ 6.9, 80.6%; 7.0 ≤ HbA1c ≤ 7.9, 90.2%; ≥8.0 HbA1c, 66.7%; P = 0.046). Patients in the HbA1c ≥ 8.0 group had significantly more tumours with vascular invasion (P = 0.041) and experienced distant metastasis significantly more often (P = 0.028) than those with other values. A multivariate analysis revealed that preoperative HbA1c ≥ 8.0 [hazard ratio (HR) 2.33; P = 0.026] and lymph node metastasis (HR 3.94; P < 0.001) were significant independent prognostic factors for recurrence.ConclusionsOur results revealed that preoperative HbA1c ≥ 8.0 is associated to poor prognosis due to the occurrence of distant metastasis and we should carefully follow these patients after surgery.Clinical registration numberHyogo Cancer Center, G-57.

Dataset Information

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis.

Publications

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets