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New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product

View Article: PubMed Central - PubMed

ABSTRACT

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig’s blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.

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The reaction pathways and analyses of peroxygenase activities of OxdAs using 1-MN as a substrate.(A) Reaction pathway for conversion of 1-MN to Russig’s blue (a), and 1-methoxy 2-naphthalenol (b). (B) The peroxygenase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) (black circles) and OxdA(H320D) (black triangles). (C) Michaelis—Menten kinetics of the peroxygenase activity of OxdAs. OxdA(WT) (a) and OxdA(H320D) (b). The reactions were carried out under the “standard assay D” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error.
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pone.0175846.g004: The reaction pathways and analyses of peroxygenase activities of OxdAs using 1-MN as a substrate.(A) Reaction pathway for conversion of 1-MN to Russig’s blue (a), and 1-methoxy 2-naphthalenol (b). (B) The peroxygenase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) (black circles) and OxdA(H320D) (black triangles). (C) Michaelis—Menten kinetics of the peroxygenase activity of OxdAs. OxdA(WT) (a) and OxdA(H320D) (b). The reactions were carried out under the “standard assay D” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error.

Mentions: Next, we examined peroxygenase activity (S + H2O2 → SO + H2O) of OxdAs (WT, H320D and H320A) in order to discover a third redox activity. To measure peroxygenase activity, we used 1-methoxynaphthalene (1-MN) as a substrate, and measured the absorbance at 610 nm due to the absorption maximum of the reaction product (Russig’s blue) [46, 47] [Fig 4A(a)]. In the reaction mixture containing 5 mM H2O2, 0.5 mM 1-MN and 1 μM OxdA, the marked production of Russig’s blue was observed under the standard assay D conditions (S9 Fig). With the use of heat-treated OxdA (for 10 min at 98°C), however, this increase in absorbance at 610 nm was not observed. These findings for the first time indicated that OxdA catalyzes a peroxygenase reaction. As for the peroxidase assays, we determined the suitable amount of H2O2 to be 5 mM for the assay (Fig 4B), by measuring each peroxygenase activity in the reaction mixture containing various concentrations of H2O2 (1–100 mM) and 0.5 mM 1-MN, this concentration giving near saturation. The suitable H2O2 concentration was different from those of the catalase and peroxidase activities (for further details, please refer to ''Discussion''). The Michaelis—Menten kinetics of the activity were determined with a suitable concentration of H2O2 [Fig 4C(a)]. The apparent Km for 1-MN and Vmax values of OxdA(WT) were found by means of Hanes-Woolf plots (S10A Fig) to be 0.070 ± 0.017 mM and 0.77 ± 0.06 units/mg, respectively (Table 4).


New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product
The reaction pathways and analyses of peroxygenase activities of OxdAs using 1-MN as a substrate.(A) Reaction pathway for conversion of 1-MN to Russig’s blue (a), and 1-methoxy 2-naphthalenol (b). (B) The peroxygenase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) (black circles) and OxdA(H320D) (black triangles). (C) Michaelis—Menten kinetics of the peroxygenase activity of OxdAs. OxdA(WT) (a) and OxdA(H320D) (b). The reactions were carried out under the “standard assay D” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5391958&req=5

pone.0175846.g004: The reaction pathways and analyses of peroxygenase activities of OxdAs using 1-MN as a substrate.(A) Reaction pathway for conversion of 1-MN to Russig’s blue (a), and 1-methoxy 2-naphthalenol (b). (B) The peroxygenase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) (black circles) and OxdA(H320D) (black triangles). (C) Michaelis—Menten kinetics of the peroxygenase activity of OxdAs. OxdA(WT) (a) and OxdA(H320D) (b). The reactions were carried out under the “standard assay D” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error.
Mentions: Next, we examined peroxygenase activity (S + H2O2 → SO + H2O) of OxdAs (WT, H320D and H320A) in order to discover a third redox activity. To measure peroxygenase activity, we used 1-methoxynaphthalene (1-MN) as a substrate, and measured the absorbance at 610 nm due to the absorption maximum of the reaction product (Russig’s blue) [46, 47] [Fig 4A(a)]. In the reaction mixture containing 5 mM H2O2, 0.5 mM 1-MN and 1 μM OxdA, the marked production of Russig’s blue was observed under the standard assay D conditions (S9 Fig). With the use of heat-treated OxdA (for 10 min at 98°C), however, this increase in absorbance at 610 nm was not observed. These findings for the first time indicated that OxdA catalyzes a peroxygenase reaction. As for the peroxidase assays, we determined the suitable amount of H2O2 to be 5 mM for the assay (Fig 4B), by measuring each peroxygenase activity in the reaction mixture containing various concentrations of H2O2 (1–100 mM) and 0.5 mM 1-MN, this concentration giving near saturation. The suitable H2O2 concentration was different from those of the catalase and peroxidase activities (for further details, please refer to ''Discussion''). The Michaelis—Menten kinetics of the activity were determined with a suitable concentration of H2O2 [Fig 4C(a)]. The apparent Km for 1-MN and Vmax values of OxdA(WT) were found by means of Hanes-Woolf plots (S10A Fig) to be 0.070 ± 0.017 mM and 0.77 ± 0.06 units/mg, respectively (Table 4).

View Article: PubMed Central - PubMed

ABSTRACT

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig’s blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.

No MeSH data available.


Related in: MedlinePlus