Evolution of two alanine glyoxylate aminotransferases in mosquito

Abstract

In the mosquito, transamination of 3-HK (3-hydroxykynurenine) to XA (xanthurenic acid) is catalysed by an AGT (alanine glyoxylate aminotransferase) and is the major branch pathway of tryptophan metabolism. Interestingly, malaria parasites hijack this pathway to use XA as a chemical signal for development in the mosquito. Here, we report that the mosquito has two AGT isoenzymes. One is the previously cloned AeHKT [Aedes aegypti HKT (3-HK transaminase)] [Han, Fang and Li (2002) J. Biol. Chem. 277, 15781-15787], similar to hAGT (human AGT), which primarily catalyses 3-HK to XA in mosquitoes, and the other is a typical dipteran insect AGT. We cloned the second AGT from Ae. aegypti mosquitoes [AeAGT (Ae. aegypti AGT)], overexpressed the enzyme in baculovirus/insect cells and determined its biochemical characteristics. We also expressed hAGT for a comparative study. The new cloned AeAGT is highly substrate-specific when compared with hAGT and the previously reported AeHKT and Drosophila AGT, and is translated mainly in pupae and adults, which contrasts with AeHKT that is expressed primarily in larvae. Our results suggest that the physiological requirements of mosquitoes and the interaction between the mosquito and its host appear to be the driving force in mosquito AGT evolution.

Scheme 1. AeAGT- and AeHKT-catalysed transamination reactions

Figure 1. The nucleotide and deduced amino acid sequences of AeAGT

Figure 2. Phylogenetic tree of AGTs

The alignment was done with CLUSTAL W in biology workbench (http://seqtool.sdsc.edu/CGI/BW.cgi#!) and the dendrogram was edited with TreeEdit (http://evolve.zoo.ox.ac.uk/software.html?id=treeedit).

Figure 3. Translational profile of AeAGT

(a) Developmental-specific expression of the AeAGT protein. An equal amount of total protein (60 μg), extracted from 1- to 5-day-old larvae (L1–L5), newly formed white pupae (P1), 12 h-old black pupae (P2) and 1-day-old female adults (A), were separated by SDS/12% PAGE and were detected on an immunoblot with anti-AeAGT polyclonal antibodies. (b) Tissue-specific expression of the AeAGT protein. Total protein was extracted from the head (H), thorax (T) and abdomen (Abd) of females at 24 h after blood feeding and an equal amount of total protein (40 μg) was resolved by SDS/12% PAGE prior to immunoblotting.

Figure 4. Verification of recombinant AeAGT by peptide MS

Purified recombinant protein was digested with trypsin, the tryptic peptides were analysed by LC–MS/MS, and collected spectral data were searched against an in-house mosquito protein database. (A) Precursor ions (MS) collected during LC–MS/MS analysis of trypsin-digested protein. Peaks in boldface are those matched to AeAGT sequence. (B) The tandem spectrum (MS/MS) of the doubly charged 714.826 peptide ion from (A) and the derived amino acid sequence. (C) Coverage map shows unambiguous match (boldface) of the spectral data to AeAGT. These data provide evidence that the expressed recombinant protein is AeAGT.

Figure 5. Transamination activity of AeAGT and hAGT towards different amino acids

Glyoxylate was used as the amino group acceptor for all amino acids, except for glycine. When glycine was used as amino group donor, pyruvate was used as an amino group acceptor. Left panel: transamination activity of AeAGT; right panel: transamination activity of hAGT.

Figure 6. Effect of pH and temperature on enzyme activity

(A, B) The activities of recombinant AeAGT and hAGT at different temperatures (A) and pH values (B). The lines with closed circles and open circles represent the specific activities of AeAGT and hAGT respectively.

Figure 7. Alignment and active site comparison of AeAGT, AnHKT, DrAGT, hAGT, AeHKT, Nostoc AGT (NosAGT) and yeast AGT (ScAGT) based on known three-dimensional structures and amino acid sequences

The residues in light or dark grey background, arbitrarily labelled P₁ to P₁₃, are PLP-binding sites; similarly, substrate-binding sites are labelled S₁ to S₇. Identity residues among all seven AGTs are labelled with a dark grey background and boxed, and similar residues are boxed only.