Purified OsDHODH activity was measured as described by Garavito et al. (2019) (link).
Three different assays were used to test the activity of the OsDHPD-T recombinant protein. In the first assay, activity was measured at 37°C, in the biological direction with 1 mM uracil as the substrate, in the presence of 150 µM NADPH or NADH, monitoring the absorbance at 340 nm, according to Yokota et al. (1994) (link). In the second assay, the reverse reaction was measured using 1 mM dihydrouracil and 150 µM NADP+ or NAD+, monitoring the increase of absorbance at 260 nm, due to the formation of uracil, according to Dolegowska et al. (2012) (link). In a third assay, normally used for measuring type I dihydroorotate dehydrogenase activity, dihydroorotate was used as the substrate, and 1 mM fumarate and 0.1 mM DCIP, following the reduction of DCIP at 600 nm, according to Zameitat et al. (2004) (link). No activity was observed for the enzyme in any of these assays.
The activity of purified OsDHP-T1 recombinant protein was assayed by measuring N-carbamoyl-ß-alanine using a colorimetric procedure (West et al., 1982 (link); Mejias-Torres & Zimmermann, 2002 (link)). Reactions were initiated by adding enzyme to concentrations of 90 - 150 nM to a solution containing 0.1 M potassium buffer, pH 8, 1 mg/mL bovine serum albumin and dihydrouracil, 37°C for 10 minutes, and then quenched followed by an incubation for 120 minutes at 70°C for color development. The concentration of ß-ureidopropionate was calculated from the absorbance at 466 nm with a standard curve. All assays were performed in triplicate, using a Beckman Coulter DU 800 UV/visible spectrophotometer.
The activity of purified Osβ-UP was measured colorimetrically for two substrates at 25°C with a modified version of the Berthelot reaction using salicylate hypochlorite (Bower and Holm-Hansen, 1980 (link)). In this assay, the NH3 produced by 100 – 200 nM enzyme in 5 – 20 minutes was converted to monochloroamine by a solution of 0.1% sodium hypochlorite in 1.5 M NaOH. Addition of a solution containing 425 mM sodium salicylate, 190 mM trisodium citrate dihydrate, 177 mM sodium potassium tartrate tetrahydrate, 0.84 mM sodium nitroprusside converted the monochloroamine to 5-aminosalicylate, which became oxidized to a blue-green dye absorbing at 650 nm. The NH3 concentration was calculated using a standard curve of NH4Cl. Absorbance was measured in microplates with a Thermo Scientific Multiskan GO UV/visible spectrophotometer.
Kinetic data for OsDHP-T1, OsßUP, and OsDHODH were fit to the Michaelis–Menten equation v = Vmax∗[S]/(Km + [S]) using GraphPad Prism v7 software.
Protein concentration was measured using the Bradford assay (Pierce) with bovine serum albumin as the standard.
Three different assays were used to test the activity of the OsDHPD-T recombinant protein. In the first assay, activity was measured at 37°C, in the biological direction with 1 mM uracil as the substrate, in the presence of 150 µM NADPH or NADH, monitoring the absorbance at 340 nm, according to Yokota et al. (1994) (link). In the second assay, the reverse reaction was measured using 1 mM dihydrouracil and 150 µM NADP+ or NAD+, monitoring the increase of absorbance at 260 nm, due to the formation of uracil, according to Dolegowska et al. (2012) (link). In a third assay, normally used for measuring type I dihydroorotate dehydrogenase activity, dihydroorotate was used as the substrate, and 1 mM fumarate and 0.1 mM DCIP, following the reduction of DCIP at 600 nm, according to Zameitat et al. (2004) (link). No activity was observed for the enzyme in any of these assays.
The activity of purified OsDHP-T1 recombinant protein was assayed by measuring N-carbamoyl-ß-alanine using a colorimetric procedure (West et al., 1982 (link); Mejias-Torres & Zimmermann, 2002 (link)). Reactions were initiated by adding enzyme to concentrations of 90 - 150 nM to a solution containing 0.1 M potassium buffer, pH 8, 1 mg/mL bovine serum albumin and dihydrouracil, 37°C for 10 minutes, and then quenched followed by an incubation for 120 minutes at 70°C for color development. The concentration of ß-ureidopropionate was calculated from the absorbance at 466 nm with a standard curve. All assays were performed in triplicate, using a Beckman Coulter DU 800 UV/visible spectrophotometer.
The activity of purified Osβ-UP was measured colorimetrically for two substrates at 25°C with a modified version of the Berthelot reaction using salicylate hypochlorite (Bower and Holm-Hansen, 1980 (link)). In this assay, the NH3 produced by 100 – 200 nM enzyme in 5 – 20 minutes was converted to monochloroamine by a solution of 0.1% sodium hypochlorite in 1.5 M NaOH. Addition of a solution containing 425 mM sodium salicylate, 190 mM trisodium citrate dihydrate, 177 mM sodium potassium tartrate tetrahydrate, 0.84 mM sodium nitroprusside converted the monochloroamine to 5-aminosalicylate, which became oxidized to a blue-green dye absorbing at 650 nm. The NH3 concentration was calculated using a standard curve of NH4Cl. Absorbance was measured in microplates with a Thermo Scientific Multiskan GO UV/visible spectrophotometer.
Kinetic data for OsDHP-T1, OsßUP, and OsDHODH were fit to the Michaelis–Menten equation v = Vmax∗[S]/(Km + [S]) using GraphPad Prism v7 software.
Protein concentration was measured using the Bradford assay (Pierce) with bovine serum albumin as the standard.
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