Analysis of the wild-type and mutant genes encoding the enzyme kynurenine monooxygenase of the yellow fever mosquito, Aedes aegypti

Abstract

Kynurenine 3-monooxygenase (KMO) catalyses the hydroxylation of kynurenine to 3-hydroxykynurenine. KMO has a key role in tryptophan catabolism and synthesis of ommochrome pigments in mosquitoes. The gene encoding this enzyme in the yellow fever mosquito, Aedes aegypti, is called kynurenine hydroxylase (kh) and a mutant allele that produces white eyes has been designated khw. A number of cDNA clones representative of wild-type and mutant genes were isolated. Sequence analyses of the wild-type and mutant cDNAs revealed a deletion of 162 nucleotides in the mutant gene near the 3'-end of the deduced coding region. RT-PCR analyses confirm the transcription of a truncated mRNA in the mutant strain. The in-frame deletion results in a loss of 54 amino acids, which disrupts a major alpha-helix and which probably accounts for the loss of activity of the enzyme. Recombinant Ae. aegypti KMO showed high substrate specificity for kynurenine with optimum activity at 40 degrees C and pH = 7.5. Kinetic parameters and inhibition of KMO activity by Cl- and pyridoxal-5-phosphate were determined.

Figure 1

Tryptophan metabolism in Aedes aegypti. The metabolic pathway in which the KMO enzyme participates starts with the conversion of tryptophan to kynurenine (left). KMO catalyses the conversion of kynurenine to 3-HK (right) that can be either converted to xanthurenic acid or oxidized to ommochrome pigments. Abbreviations: 3-HK, 3-hydroxykynurenine; KMO, kynurenine monooxygenase.

Figure 2

Multiple alignment of the amino acid sequence of AeKMO with KMOs from diverse animals. AE, AeKMO (GenBank accession no. AF325508); DM, Drosophila melanogaster KMO (GenBank accession no. U56245); BM, Bombyx mori KMO (GenBank accession no. AB063490); RN, Rattus norvegicus KMO (GenBank accession no. AF056031); SS, Sus scrofa KMO (GenBank accession no. AF163971); HS, Homo sapiens KMO (GenBank accession no. Y13153). The white letters with black background indicate identical amino acids. The underlined sequences are those used to design degenerate primers. The first boxed fragment is the dinucleotide-binding motif of ‘xhxhGxGxxGxxxhxxh(x)₈hxhE(D)’, where x is any residue and h is a hydrophobic residue. The second boxed fragment is a putative consensus domain for both FAD and NAD(P)H binding.

Figure 3

Comparisons of the predicted KMO proteins of the Rockefeller, kh^w and Liverpool strains. (A) Amino acid alignment of the predicted KMO proteins. The Rockefeller (R) and kh^w (k) proteins are identical except for a fifty-four amino acid deletion denoted by the dashes (−). The Liverpool (L) and Rockefeller have eight different amino acids indicated in bold (GenBank accession no. Rockefeller AY194225; kh^w AY194224). (B) The large deletion in the kh^w KMO disrupts the α-helixal structure in the carboxyl terminal region of the protein. The predicted α-helicies are shown in red and the deletion as a cross-hatched box. The Garnier–Robson model (Persson, 2000) was used to predict the structure.

Figure 4

Developmental expression profiles of wild-type and mutant genes. RT-PCR analyses of total RNA from first (L₁), second (L₂), third (L₃) and fourth (L₄) larval instars, early (P₁) and late-stage (P₂) pupae, adult males (M), and sugar-(F_sf) and blood-fed (F_bf), adult females from the wild-type strain, Rockefeller (Rock), and the mutant kh^w strain.

Figure 5

Purification of rAeKMO. Lane 1 shows the SDS-PAGE protein profiles of the supernatant of cell lysate obtained from uninfected Sf9 cells; Lane 2 shows the protein profiles of the supernatant of cell lysate obtained from Sf9 cells infected with an AeKMO recombinant virus; Lane 3 shows the purified rAeKMO; and Lane 4 shows the protein molecular mass standards on the same polyacrylamide gel.

Figure 6

Effects of temperature and pH on rAeKMO activity. The detailed assay methods are described in the Experimental procedures. The biochemical activity was assayed with kynurenine (2 mM) as a hydroxyl group acceptor and NADPH (0.8 mM) as an electron donor. (A) rAeKMO activity at temperatures from 35 to 70 °C; (b) the pH profile of rAeKMO, analysed using Enzyme Kinetics Module (SPSS Science).

Figure 7

Inhibition of KMO by PLP. The Michaelis–Menten curves of different concentrations of PLP: zero (closed circles), 0.05 mM (open circles), 0.1 mM (closed triangles) and 0.2 mM (open triangles). (A) Activity was assayed at a fixed concentration of NADPH (3.2 mM) and different concentrations of kynurenine (0.125, 0.25, 0.5, 1, 2, 4 mM); (B) activity was assayed at a fixed concentration of kynurenine (4 mM) and different concentrations of NADPH (0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM).

Figure 8

Inhibition of KMO by Cl⁻¹. The Michaelis–Menten curves of different concentrations of Cl⁻¹: zero (closed circles), 25 mM (open circles), 50 mM (closed triangles), 100 mM (open triangles) and 200 mM (closed squares). (A) Activity was assayed at a fixed concentration of NADPH (3.2 mM) and different concentrations of kynurenine (0.125, 0.25, 0.5, 1, 2, 4 mM); (B) activity was assayed at a fixed concentration of kynurenine (4 mM) and different concentrations of NADPH (0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM).