Comparative characterization of Aedes 3-hydroxykynurenine transaminase/alanine glyoxylate transaminase and Drosophila serine pyruvate aminotransferase

Abstract

This study describes the comparative analysis of two insect recombinant aminotransferases, Aedes aegypti 3-hydroxykynurenine (3-HK) transaminase/alanine glyoxylate aminotransferase (Ae-HKT/AGT) and Drosophila melanogaster serine pyruvate aminotransferase (Dm-Spat), which share 52% identity in their amino acid sequences. Both enzymes showed AGT activity. In addition, Ae-HKT/AGT is also able to catalyze the transamination of 3-HK or kynurenine with glyoxylate, pyruvate or oxaloacetate as the amino acceptor. Kinetic analysis and other data suggest that Ae-HKT/AGT plays a critical role in mosquito tryptophan catabolism by detoxifying 3-HK and that Dm-Spat is primarily involved in glyoxylate detoxification.

Fig. 1

Recombinant Ae-HKT/AGT and Dm-Spat expression and purification. Soluble proteins were obtained as described in Section 2 and subjected to SDS–PAGE analysis. Lanes (left side) illustrate protein profiles of crude soluble proteins from cells infected with the Ae-HKT/AGT recombinant virus (A1), the remaining proteins after DEAE Sepharose (A2), phenyl Sepharose (A3) and hydroxyapatite (A4) chromatographic separations, respectively. Lanes (right side) represent soluble protein from cells infected with the Dm-Spat recombinant virus (B1), the remaining protein after phenyl Sepharose (B2) and hydroxyapatite (B3) chromatographic separations, respectively. Lane A shows the protein molecular mass standards.

Fig. 2

Relative molecular mass (M_r) of native Ae-HKT/AGT and Dm-Spat. The M_r of native Ae-HKT/AGT and Dm-AGT was assessed by gel filtration chromatography on a Pharmacia Superdex 200 HR gel filtration column (1×30 cm) with thyroglobulin (670 000), γ-globulin (158 000), ovalbumin (44 000) and myoglobin (17 000) (Bio-Rad) as molecular mass standards. The mobile phase consisted of 20 mM phosphate buffer (pH 7.0) containing 150 mM NaCl at a flow rate of 0.2 ml per minute. Under the applied chromatographic conditions, the retention times of thyroglobulin (F), γ-globulin (E), Ae-HKT/AGT (D), Dm-Spat (C), ovalbumin (B), and myoglobin (A) were 44.3, 58.8, 60.0, 65.3, 77.4 and 84.0 min, respectively.

Fig. 3

Effect of pH and temperature on enzyme activity. Assay conditions were the same as described in Section 2, except that glyoxylate was used as the amino acceptor and potassium phosphate buffer was used at pH 6–8, borate buffer at pH 9, and carbonate buffer at pH 10. The effect of temperature on transamination activity was determined by incubation of the corresponding substrate preparations at 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C or 90°C for 5 min prior to the addition of the enzyme and continuously incubated for 5 min following enzyme addition. Panels A and B show the activities of recombinant Ae-HKT/AGT and Dm-Spat under different temperatures and pHs, respectively. The lines with closed circles, closed triangles and open circles represent the specific activities of Dm-Spat towards alanine, Ae-HKT/AGT towards 3-HK, and Ae-HKT/AGT towards alanine, respectively.