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

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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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Michaelis—Menten kinetics of the catalase activity of OxdAs.The reactions were carried out under the “standard assay A” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error. OxdA(WT) (A), OxdA(H320D) (B) and OxdA(H320A) (C).
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pone.0175846.g001: Michaelis—Menten kinetics of the catalase activity of OxdAs.The reactions were carried out under the “standard assay A” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error. OxdA(WT) (A), OxdA(H320D) (B) and OxdA(H320A) (C).

Mentions: OxdA catalyzes the dehydration of aldoximes to the corresponding nitriles, but not redox reactions. First, we examined the catalase activity of OxdA by observing the increase in O2 by using an oxygen electrode. In the reaction mixture containing 10 mM H2O2 and 2 μM OxdA, the production of O2 (S1A Fig) was detected. On the other hand, an increase in O2 was not detected in the reaction mixture containing heat-treated OxdA (for 10 min at 98°C), which did not give the specific absorption peak of heme (S2A Fig). These findings for the first time indicated that OxdA catalyzes a catalase reaction. Then, we determined the Michaelis—Menten kinetics of the activity at the following concentrations of H2O2 (10–100 mM) (Fig 1A). The apparent Km and Vmax values of OxdA were found by means of Hanes-Woolf plots (S3A Fig) to be 22 ± 4 mM and 1.9 ± 0.1 units/mg, respectively (Table 1).


New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product
Michaelis—Menten kinetics of the catalase activity of OxdAs.The reactions were carried out under the “standard assay A” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error. OxdA(WT) (A), OxdA(H320D) (B) and OxdA(H320A) (C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5391958&req=5

pone.0175846.g001: Michaelis—Menten kinetics of the catalase activity of OxdAs.The reactions were carried out under the “standard assay A” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error. OxdA(WT) (A), OxdA(H320D) (B) and OxdA(H320A) (C).
Mentions: OxdA catalyzes the dehydration of aldoximes to the corresponding nitriles, but not redox reactions. First, we examined the catalase activity of OxdA by observing the increase in O2 by using an oxygen electrode. In the reaction mixture containing 10 mM H2O2 and 2 μM OxdA, the production of O2 (S1A Fig) was detected. On the other hand, an increase in O2 was not detected in the reaction mixture containing heat-treated OxdA (for 10 min at 98°C), which did not give the specific absorption peak of heme (S2A Fig). These findings for the first time indicated that OxdA catalyzes a catalase reaction. Then, we determined the Michaelis—Menten kinetics of the activity at the following concentrations of H2O2 (10–100 mM) (Fig 1A). The apparent Km and Vmax values of OxdA were found by means of Hanes-Woolf plots (S3A Fig) to be 22 ± 4 mM and 1.9 ± 0.1 units/mg, respectively (Table 1).

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