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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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Analyses of peroxidase activities of OxdAs in the presence of ABTS.(A) The peroxidase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) [(a) black circles], OxdA(H320D) [(a) black triangles], and OxdA(H320A) [(b) black square]. (B) Michaelis—Menten kinetics of the peroxidase activity of OxdAs. OxdA(WT) (a), OxdA(H320D) (b) and OxdA(H320A) (c). The reactions with ABTS as a substrate were carried out under the “standard assay C” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error.
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pone.0175846.g003: Analyses of peroxidase activities of OxdAs in the presence of ABTS.(A) The peroxidase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) [(a) black circles], OxdA(H320D) [(a) black triangles], and OxdA(H320A) [(b) black square]. (B) Michaelis—Menten kinetics of the peroxidase activity of OxdAs. OxdA(WT) (a), OxdA(H320D) (b) and OxdA(H320A) (c). The reactions with ABTS as a substrate were carried out under the “standard assay C” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error.

Mentions: (ii) Instead of guaiacol, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) was used as a substrate. With the reaction mixture consisting of 25 mM H2O2, 2.5 mM ABTS and 1 μM OxdA(WT) under the standard assay C conditions (S7 Fig), we observed an increase in the absorbance at 430 nm due to the absorption maximum for a peroxidase reaction product (ABTS radical) [45]. As for the peroxidase assay with guaiacol as a substrate, the peroxidase activity in the reaction mixture containing various concentrations of H2O2 (1~100 mM) and excess ABTS (2.5 mM) for OxdA was measured [Fig 3A(a)]. Even with 100 mM H2O2, surprisingly, the peroxidase activity using ABTS as a substrate was inhibited, indicating the suitable concentration of H2O2 was 25 mM for the assay. The tendency of H2O2 concentration-dependent inhibition was different from those for the catalase (S5 Fig) and peroxidase (using guaiacol as a substrate) (Fig 2A) reactions. Inhibition of the peroxidase activity (using ABTS as a substrate) by a low concentration of H2O2 would be caused by competition between the catalase and peroxidase activities (for further details, please refer to ''Discussion''). On determination of the Michaelis—Menten kinetics of the activity in the reaction mixture containing 25 mM H2O2 [Fig 3B(a)], the apparent Km for ABTS and Vmax values of OxdA(WT) were found by means of Hanes-Woolf plots (S8A Fig) to be 0.21 ± 0.04 mM and 3.4 ± 0.2 units/mg, respectively (Table 3). Furthermore, we succeeded in the detection of peroxidase activity of mutant OxdAs (H320D and H320A) (S7 Fig). The suitable concentrations of H2O2 for OxdAs (H320D and H320A) were determined to be 25 and 10 mM for the peroxidase assay (Fig 3A), respectively, and the kinetic parameters were determined by means of Hanes-Woolf plots (S8 and S8C Fig) to be Km values 0.12 ± 0.02 and 0.21 ± 0.03 mM for ABTS, and Vmax values 33 ± 2 and 1.3 ± 0.1 units/mg, respectively [Fig 3B(b) and 3Bc)] (Table 3). These findings demonstrated that the Vmax value (33 ± 2 units/mg) of OxdA(H320D) was about 10 times higher than that (3.4 ± 0.2 units/mg) of OxdA(WT), while that of OxdA(H320A) (1.3 ± 0.1 units/mg) was about 3 times lower than that of OxdAs (WT). On the other hand, the Km value (0.12 ± 0.02 mM) of OxdA(H320D) for ABTS was nearly half of those (0.21 ± 0.04 and 0.21 ± 0.03 mM) of OxdAs (WT and H320A).


New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product
Analyses of peroxidase activities of OxdAs in the presence of ABTS.(A) The peroxidase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) [(a) black circles], OxdA(H320D) [(a) black triangles], and OxdA(H320A) [(b) black square]. (B) Michaelis—Menten kinetics of the peroxidase activity of OxdAs. OxdA(WT) (a), OxdA(H320D) (b) and OxdA(H320A) (c). The reactions with ABTS as a substrate were carried out under the “standard assay C” 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.g003: Analyses of peroxidase activities of OxdAs in the presence of ABTS.(A) The peroxidase activities of OxdAs depending on the H2O2 concentration. OxdA(WT) [(a) black circles], OxdA(H320D) [(a) black triangles], and OxdA(H320A) [(b) black square]. (B) Michaelis—Menten kinetics of the peroxidase activity of OxdAs. OxdA(WT) (a), OxdA(H320D) (b) and OxdA(H320A) (c). The reactions with ABTS as a substrate were carried out under the “standard assay C” conditions as described under “Materials and Methods.” For all data points, values are means ± mean error.
Mentions: (ii) Instead of guaiacol, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) was used as a substrate. With the reaction mixture consisting of 25 mM H2O2, 2.5 mM ABTS and 1 μM OxdA(WT) under the standard assay C conditions (S7 Fig), we observed an increase in the absorbance at 430 nm due to the absorption maximum for a peroxidase reaction product (ABTS radical) [45]. As for the peroxidase assay with guaiacol as a substrate, the peroxidase activity in the reaction mixture containing various concentrations of H2O2 (1~100 mM) and excess ABTS (2.5 mM) for OxdA was measured [Fig 3A(a)]. Even with 100 mM H2O2, surprisingly, the peroxidase activity using ABTS as a substrate was inhibited, indicating the suitable concentration of H2O2 was 25 mM for the assay. The tendency of H2O2 concentration-dependent inhibition was different from those for the catalase (S5 Fig) and peroxidase (using guaiacol as a substrate) (Fig 2A) reactions. Inhibition of the peroxidase activity (using ABTS as a substrate) by a low concentration of H2O2 would be caused by competition between the catalase and peroxidase activities (for further details, please refer to ''Discussion''). On determination of the Michaelis—Menten kinetics of the activity in the reaction mixture containing 25 mM H2O2 [Fig 3B(a)], the apparent Km for ABTS and Vmax values of OxdA(WT) were found by means of Hanes-Woolf plots (S8A Fig) to be 0.21 ± 0.04 mM and 3.4 ± 0.2 units/mg, respectively (Table 3). Furthermore, we succeeded in the detection of peroxidase activity of mutant OxdAs (H320D and H320A) (S7 Fig). The suitable concentrations of H2O2 for OxdAs (H320D and H320A) were determined to be 25 and 10 mM for the peroxidase assay (Fig 3A), respectively, and the kinetic parameters were determined by means of Hanes-Woolf plots (S8 and S8C Fig) to be Km values 0.12 ± 0.02 and 0.21 ± 0.03 mM for ABTS, and Vmax values 33 ± 2 and 1.3 ± 0.1 units/mg, respectively [Fig 3B(b) and 3Bc)] (Table 3). These findings demonstrated that the Vmax value (33 ± 2 units/mg) of OxdA(H320D) was about 10 times higher than that (3.4 ± 0.2 units/mg) of OxdA(WT), while that of OxdA(H320A) (1.3 ± 0.1 units/mg) was about 3 times lower than that of OxdAs (WT). On the other hand, the Km value (0.12 ± 0.02 mM) of OxdA(H320D) for ABTS was nearly half of those (0.21 ± 0.04 and 0.21 ± 0.03 mM) of OxdAs (WT and H320A).

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