Crystal Structures of Aedes aegypti Alanine Glyoxylate Aminotransferase

Jianyong Li,Scott R. Wilson,Nancy Vogelaar,Yi Gui Gao,Qian Han,Howard Robinson,Menico Rizzi

doi:10.1074/jbc.m607032200

Abstract

Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75A high-resolution three-dimensional crystal structure of AGT from the mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1A resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.

Highlights

Mosquitoes transmit malaria, which is considered the most prevalent life-threatening disease in the world [1]
Overall Structure—The structure of Aedes aegypti (AeAGT) was determined by molecular replacement and refined to 1.75 Å resolution
The protein architecture revealed by AeAGT consists of the prototypical fold of aminotransferase subgroup I [26, 27], characterized by an N-terminal arm, which consists of a random coiled stretch made of residues 1–18, a small C-terminal domain, and a large N-terminal domain (Fig. 1), which is similar to the reported AGT structures from other species [14, 17, 28, 29]

Summary

Crystal Structures of Aedes aegypti Alanine Glyoxylate Aminotransferase*

Because mosquitoes cannot dispose of 3-HK through hydrolysis and subsequent oxidations like mammals, the transamination of the chemically reactive 3-HK to the relatively chemically stable XA by HKT is considered the mechanism by which mosquitoes prevent 3-HK from overaccumulating [7]. Such an explanation remains a valid argument for the functional evolution of the mosquito AGTs. Interestingly, 3-HK is the initial precursor for the production of ommochromes that are major eye pigments in mosquitoes. Glyoxylate aminotransferase; AnHKT, Anopheles HKT; DrAGT, Drosophila AGT; hAGT, human AGT; HKT, 3-HK transaminase; LLP, lysine-pyridoxal 5-phosphate; PLP, pyridoxal 5-phosphate; XA, xanthurenic acid

Space group

Crystal data

Biochemical characterization

Overexpression and Purification of

RESULTS AND DISCUSSION

Rizzi and Jianyong Li