Motif‐based search for a novel fructosyl peptide oxidase from genome databases

Kim, Seungsu; Ferri, Stefano; Tsugawa, Wakako; Mori, Kazushige; Sode, Koji

doi:10.1002/bit.22710

Cited by 19 publications

(11 citation statements)

References 46 publications

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“…The flavin ring is shown for reference moiety of the ligand, although the sugar moiety presents a higher degree of mobility within the binding site of FPOX-E. Conversely, f-val shows a primary cluster that is more populated (67 % and 73 % of the time in FPOX-E and PnFPOX, respectively), which suggests that, overall, these enzymes have a higher affinity for f-val. This observation correlates well with the experimentally determined enzymatic activity of PnFPOX [24], while the enzymatic characterization of FPOX-E is incomplete [25]. Finally, we observe that both f-lys and f-val are highly unstable in the N1-1-FAOD enzyme, as suggested by the low population of the major conformational cluster (46 % for f-lys and 18 % for f-val) and by the high mobility of the substrates in the binding pocket, also involving the otherwise usually stable sugar moiety.…”

Section: Enzyme Structures and Sequence Comparisonssupporting

confidence: 89%

Molecular Dynamics Simulations Provide Insights into Structure and Function of Amadoriase Enzymes

Rigoldi¹,

Spero²,

Vedove³

et al. 2017

Research Bulletin of the National Technical University of Ukraine "Kyiv Politechnic Institute"

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Background. Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. Objective. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnel, ligand binding residues and ligand binding energies. Methods. We used a combination of homology modeling and molecular dynamics simulations to provide insights into the structural difference between the five enzymes of the FAOX family. Results. We first predicted the structure of the N1-1-FAOD and PnFPOX enzymes using homology modelling. Then, we used these models and the experimental crystal structures of Amadoriase I, Amadoriase II and FPOX-E to run extensive molecular dynamics simulations in order to compare the structures of these FAOX enzymes and assess their relevant interactions with two relevant ligands, f-val and f-lys. Conclusions. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels.

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Section: Enzyme Structures and Sequence Comparisonssupporting

confidence: 89%

Molecular Dynamics Simulations Provide Insights into Structure and Function of Amadoriase Enzymes

Rigoldi¹,

Spero²,

Vedove³

et al. 2017

Research Bulletin of the National Technical University of Ukraine "Kyiv Politechnic Institute"

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“…Kim et al . reported that G60 of PnFPOX is the decisive residue for F-VH oxidation activity 19 . Seventy-first residue and 59 residue (corresponding to G60 of PnFPOX) of AnFPOX-15 were structurally distant, as 71 residue is located in helix 2 and behind loop A, whereas 59 residue is located in loop A.…”

Section: Resultsmentioning

confidence: 99%

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

Ogawa

Kimura

Umehara

et al. 2019

Sci Rep

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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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“…Fructosylvalylhistidine (Fru-ValHis) is released from HbA1c while fructosyl-lysine (Fru-Lys) is released from glycated albumin. Fru-ValHis and Fru-Lys can be considered as model compounds for HbA1c and glycated albumin, respectively (Kim et al 2010). However, wild-type FPOX has activities toward both Fru-ValHis and Fru-Lys.…”

Section: Introductionmentioning

confidence: 99%

Engineering an efficient mutant of Eupenicillium terrenum fructosyl peptide oxidase for the specific determination of hemoglobin A1c

Shahbazmohammadi

Sardari

Lari

et al. 2019

Appl Microbiol Biotechnol

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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.

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Motif‐based search for a novel fructosyl peptide oxidase from genome databases

Cited by 19 publications

References 46 publications

Molecular Dynamics Simulations Provide Insights into Structure and Function of Amadoriase Enzymes

Molecular Dynamics Simulations Provide Insights into Structure and Function of Amadoriase Enzymes

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

Engineering an efficient mutant of Eupenicillium terrenum fructosyl peptide oxidase for the specific determination of hemoglobin A1c

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