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Originally published In Press as doi:10.1074/jbc.M201202200 on March 5, 2002

J. Biol. Chem., Vol. 277, Issue 18, 15781-15787, May 3, 2002
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3-Hydroxykynurenine Transaminase Identity with Alanine Glyoxylate Transaminase

A PROBABLE DETOXIFICATION PROTEIN IN AEDES AEGYPTI*

Qian Han, Jianmin Fang, and Jianyong LiDagger

From the Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802

Received for publication, February 5, 2002, and in revised form, February 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study describes the functional characterization of a specific mosquito transaminase responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). The enzyme was purified from Aedes aegypti larvae by ammonium sulfate fractionation, heat treatment, and various chromatographic techniques, plus non-denaturing electrophoresis. The purified transaminase has a relative molecular mass of 42,500 by SDS-PAGE. N-terminal and internal sequencing of the purified protein and its tryptic fragments resolved a partial N-terminal sequence of 19 amino acid residues and 3 partial internal peptide sequences with 7, 10, and 7 amino acid residues. Using degenerate primers based on the partial internal sequences for PCR amplification and cDNA library screening, a full-length cDNA clone with a 1,167-bp open reading frame was isolated. Its deduced amino acid sequence consists of 389 amino acid residues with a predicted molecular mass of 43,239 and shares 45-46% sequence identity with mammalian alanine glyoxylate transaminases. Northern analysis shows the active transcription of the enzyme in larvae and developing eggs. Substrate specificity analysis of this mosquito transaminase demonstrates that the enzyme is active with 3-HK, kynurenine, or alanine substrates. The enzyme has greater affinity and catalytic efficiency for 3-HK than for kynurenine and alanine. The biochemical characteristics of the enzyme in conjunction with the profiles of 3-HK transaminase activity and XA accumulation during mosquito development clearly point out its physiological function in the 3-HK to XA pathway. Our data suggest that the mosquito transaminase was evolved in a manner precisely reflecting the physiological requirement of detoxifying 3-HK produced in the tryptophan oxidation pathway in the mosquito.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

3-HK1 is a natural metabolite in the tryptophan oxidation pathway. However, it is oxidized easily, stimulating the production of reactive oxygen species (1-5). 3-HK concentration is elevated in the brains of patients with AIDS-related dementia (6), Huntington's disease (7, 8), and hepatic encephalopathy (9). Recently it has been reported that 3-HK, at low micromolar concentrations, induces apoptosis of neurons prepared from the rat striatum (3, 4, 10). In insects, injection of 3-HK into adult Neobellieria bullata caused instant paralysis, which was followed by death (11). 3-HK also is present in the mammalian lens, where its oxidation product might cross-link lens proteins and contribute to nuclear cataract formation (12-15). To maintain physiological conditions, it apparently is critical for living organisms to be able to tightly regulate the level of 3-HK, thereby preventing overaccumulation of 3-HK.

Although 3-HK is toxic to living organisms, the tryptophan to 3-HK pathway is actually the major branch pathway of tryptophan catabolism in mammals. Fortunately, mammals have kynureninase that can efficiently hydrolyze 3-HK to alanine and 3-hydroxyanthranilic acid, and the latter can be eventually oxidized to CO2 and H2O or used to synthesize NAD+ and NADP+ through a quinolinic acid intermediate (16). The mammalian kynureninase is also capable of catalyzing the hydrolysis of kynurenine, but it has a much higher affinity for 3-HK than kynurenine (17), suggesting that the enzyme may play a major role in preventing the accumulation of 3-HK in mammals. Results from our previous study demonstrate that oxidation of tryptophan to 3-HK is also a major pathway for tryptophan catabolism in Aedes aegypti mosquitoes, especially during larval and egg development (18). However, in contrast to mammals, mosquitoes cannot hydrolyze 3-HK and kynurenine because of the lack of kynureninase, which prevents both kynurenine and 3-HK from being completely oxidized or used for NAD+ and NADP+ synthesis. As a result, mosquitoes must deal with 3-HK in a different manner.

Our studies indicate that mosquitoes have an efficient mechanism for controlling the level of 3-HK through the conversion of the chemically reactive and potentially toxic 3-HK to a chemically stable XA by transaminase-mediated reactions. Therefore, the 3-HK to XA pathway is considered an essential detoxification pathway in mosquitoes. Crude protein extracted from A. aegypti larvae has a fairly high 3-HK transamination activity, but activity diminished once the larvae metamorphosed to pupae (18). The 3-HK transamination activity profile also correlates well with levels of XA accumulation in A. aegypti during development (18). It seems clear that a transaminase more specific for the transamination of 3-HK is present in A. aegypti and is responsible for preventing 3-HK accumulation in mosquitoes during development. Based on the reaction it catalyzes, we termed the enzyme 3-HK transaminase (HKT) in our previous report (18).

In mammals, kynurenine aminotransferase (KAT) catalyzes the transamination of kynurenine and 3-HK to kynurenic acid (KA) and XA, respectively (19-23). Crude mosquito larval protein, although more active to 3-HK, was capable of catalyzing the transamination of kynurenine to kynurenic acid; therefore, we presumed that the mosquito HKT might actually be quite similar to mammalian KAT. Recently, we isolated the mosquito KAT clone (GenBankTM accession number AF395204) and expressed it in a baculovirus/insect expression system. The mosquito KAT shares about 50% sequence identity to mammalian KATs, and its recombinant protein is capable of catalyzing the transamination of kynurenine to kynurenic acid. However, in contrast to mammalian KATs, the expressed protein showed no activity for 3-HK (24). It seems that a different transaminase must be responsible for catalyzing the transamination of 3-HK to XA in mosquitoes.

XA has been reported to induce gametogenesis in Plasmodium (malaria parasites) (25), and it has been suggested to be the inducer of Plasmodium development in the mosquito (26). The accumulation of XA and its function on Plasmodium development in the mosquito correlates with the fact that the mosquito hosts Plasmodium as its biological transmission vector. HKT may be a target gene for the study of the parasite-host relationship between malaria and mosquito, and the result may be beneficial to malaria control.

To understand the specific transaminase responsible for catalyzing the transamination of 3-HK to XA in mosquitoes, we purified the mosquito HKT, obtained its partial internal amino acid sequences, and isolated its cDNA based on the partial sequence data. A BLAST search against NCBI databases revealed that the mosquito transaminase shared 45-46% sequence identity to mammalian alanine glyoxylate transaminases (AGTs). The high sequence similarity of the proposed mosquito HKT with AGTs raises a fundamental question regarding the identity of this mosquito transaminase. With the purified enzyme in this study, we were able to critically characterize the mosquito transaminase. Our data suggest that the primary function of this mosquito transaminase is the transamination of 3-HK to XA, and the enzyme plays an important physiological role in mosquitoes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

Chemicals were purchased from Sigma unless otherwise specified. Protein molecular weight markers, DEAE-Sepharose, phenyl-Sepharose, hydroxyapatite, and UNO-Q ion-exchange columns (12 × 53 mm and 7 × 35 mm) were obtained from Bio-Rad. A C18 reversed phase column (250 × 4.6 mm; particle size, 5 µm; pore size, 300 Å) was from Vydac (Hesperia, CA).

Larval Crude Protein Isolation

Mosquitoes (A. aegypti black-eyed Liverpool strain) used in this study were reared according to a previously described method (27). About 500 g (wet weight) of 5-day-old larvae were collected and homogenized in a Palytron homogenizer (England) in 20 mM phosphate buffer, pH 6.2, containing 40% ammonium sulfate, 0.05 mM pyridoxal phosphate, 10 mM beta -ME, 1 mM phenylmethylsulfonyl fluoride, and 1 mM phenylthiourea. The homogenate was centrifuged at 18,000 × g for 10 min, and the pellets were discarded. Solid (NH4)2SO4 was added to the supernatant to 60% saturation with gentle stirring. Precipitated proteins were separated by centrifugation (18,000 × g for 10 min) and re-dissolved in 20 mM phosphate buffer, pH 6.2, containing 10 mM beta -ME, 0.05 mM PLP. The dissolved protein sample was heated to 55 °C for 5 min, chilled on ice for 15 min, and centrifuged (18,000 × g for 10 min) to obtain supernatant that was used as the starting material for HKT purification.

Chromatographic Purification

Phenyl-Sepharose Chromatography-- Supernatant from the heat-treated protein sample, which contained the major HKT activity, was loaded to a phenyl-Sepharose column (5 × 12 cm) equilibrated with 20 mM phosphate buffer, pH 6.2, containing 1 M (NH4)2SO4, 10 mM beta -ME, and 0.05 mM PLP. Proteins were eluted using a decreasing linear (NH4)2SO4 gradient from 1 to 0.05 M prepared in 10 mM phosphate buffer, pH 6.2, containing 10 mM beta -ME and 0.05 mM PLP. It was found that both PLP and beta -ME were effective in protecting HKT from inactivation. The 10 mM phosphate buffer, pH 6.2, containing 0.05 mM PLP and 10 mM beta -ME, which was extensively used during chromatographic separations of HKT, was defined as buffer A.

DEAE-Sepharose Chromatography-- The eluted HKT-active fractions in buffer A were directly applied to a DEAE-Sepharose column (2.5 × 12 cm) equilibrated with buffer A. The enzyme was eluted by a linear KCl gradient from 0 to 300 mM prepared in 500 ml of buffer A.

Hydroxyapatite and UNO-Q Column Chromatography-- Active fractions from DEAE-Sepharose chromatography were combined and directly applied to a hydroxyapatite column (1 × 12 cm) equilibrated with buffer A. Proteins were eluted by a linear phosphate gradient from 10 to 400 mM prepared in buffer A at a flow rate of 1.5 ml min-1 during a 50-min period. Active fractions were pooled, concentrated, and exchanged with buffer A using a Centriprep YM-30 concentrator (Millipore). The concentrated HKT-active sample (1.5 ml) was applied to an UNO-Q ion-exchange column (12 × 53 mm) and eluted by a linear KCl gradient from 10 to 300 mM prepared in buffer A with a flow rate of 1 ml min-1 during a 50-min period. Active fractions were pooled, concentrated, and exchanged with buffer A using Centriprep YM-30 concentrator (Millipore). The concentrated and buffer-exchanged HKT fraction was applied to the second hydroxyapatite column (7 × 52 mm), and proteins were eluted by a linear phosphate gradient from 10 to 400 mM prepared in buffer A at a flow rate of 0.5 ml min-1 during a 50-min period. The pooled fractions with HKT activity were concentrated to 0.2 ml with a Centricon YM-30 concentrator (Millipore), rechromatographed on the second UNO-Q column (7 × 35 mm), and eluted by a linear KCl gradient from 0 to 300 mM prepared in buffer A with a flow rate of 0.5 ml min-1 during a 30-min period. The HKT-active fractions were pooled and concentrated.

Preparative Electrophoresis

200 µl of concentrated HKT fractions from the second UNO-Q chromatography were mixed with 50 µl of sample buffer containing 25 µl of 200 mM Tris buffer (pH 8.8) and 25 µl of glycerol, with a minimum amount of bromphenol blue for visualization. The mixture was loaded into a preparative well of a non-denaturing linear gradient (5-20%) polyacrylamide gel. Electrophoresis was conducted at a constant voltage of 50 V for 12 h at 4 °C. After electrophoresis, the gel was sectioned into horizontal strips (2-mm wide). A small piece (2 × 2 mm) was cut from individual strips and mixed with 50 µl of a substrate preparation containing 10 mM 3-HK, 10 mM pyruvate, and 40 µM PLP prepared using 200 mM phosphate buffer, pH 7.0, for the HKT activity assay. After the HKT-active gel strip was identified, protein in the gel strip was electroeluted using a Centrieluter (Millipore) and concentrated using a Centricon YM-30 concentrator (Millipore). The concentrated protein was used for biochemical characterizations, protein sequencing by Edman degradation, and analysis for purity and relative molecular mass by SDS-PAGE and reversed phase chromatography (28).

N-terminal and Internal Sequencing

The HKT-active protein after ND-PAGE was further separated by SDS-PAGE, and the protein was electroblotted on a polyvinylidene difluoride membrane for N-terminal sequencing by Edman degradation using a Procise 494 protein sequencer at the University of Illinois Biotechnology Center. To obtain partial internal sequences, the ND-PAGE-derived HKT fraction was chromatographed by HPLC on a C18 reversed phase column (4.6 × 250 mm) to separate contaminants remaining in the enzyme fraction. The HKT peak was collected, lyophilized, and digested using trypsin. The trypsin digest was separated by capillary HPLC, and three well resolved peptide fragments were collected directly on polyvinylidene difluoride membranes for Edman digestion.

cDNA Cloning

PCR Amplification-- A forward degenerated primer (5'-GCNATHAAYATGGCNAC-3') and a reversed degenerate primer (5'-GCTTNACRAADATYTC-3') were designed based on internal amino acid sequences (AINMAT and EIFVK, respectively) and used for PCR amplification of the first strand cDNA synthesized from total RNA of 3-day-old A. aegypti larvae using a cDNA synthesizing kit (Invitrogen). A 600-bp fragment was amplified, cloned into a PCR2.1-TOPO TA cloning vector (Invitrogen), and then sequenced using the BigDyeTM Terminator Cycle sequencing kit (Applied Biosystems). The deduced amino acid sequence contained the partial internal peptide sequences used to design degenerate primers and also included the other partial internal amino acid residues (VLEGPADKPF) not used for primer design, indicating the amplified DNA fragment was a partial cDNA of the purified HKT.

cDNA Cloning and Sequencing-- An A. aegypti larval cDNA library was constructed using a library construction kit (Stratagene) according to the manufacturer's instructions. The 600-bp fragment was labeled with [alpha -32P]dCTP (PerkinElmer Life Sciences) and used for cDNA library screening. A total of 5 × 105 plaques were screened at high stringency. Ten clones were isolated and three of them were sequenced. Sequence assembly was carried out with SeqEdit software (V1.0.3). Sequence data were analyzed using the Biology WorkBench 3.2 program (workbench.sdsc.edu) and the software package from the Genetics Computer Group, Inc. (GCG, University of Wisconsin, Madison).

Northern Hybridization

Total RNA was isolated (using Trizol reagent, Invitrogen) from larvae at 1-6 days after hatching, from pupae at 0.5 and 12 h after pupation, and from ovaries collected at 24 h postbloodfeeding. Total RNA was electrophoresed in a 1% agarose-formaldehyde gel in 20 mM MOPS buffer containing 4 mM sodium acetate and 1 mM EDTA at 5 V/cm for 3 h. RNA was transferred to a positive charge nylon membrane (Ambion) and cross-linked using a Bio-Rad UV cross-linker. The blot was hybridized sequentially with the [32P]dCTP-labeled 600-bp HKT fragment and then with a 500-bp probe generated from 18 S ribosomal RNA of A. aegypti as a loading control. After hybridization at 42 °C for 16 h, the blots were washed with increasing stringency (twice in 2× SSC containing 0.1% SDS at room temperature for 25 min and twice with 0.1× SSC containing 0.1% SDS at 68 °C for 30 min) and exposed to x-ray films at -80 °C.

Biochemical Characterizations

Substrate Specificity and Kinetic Analysis-- During enzyme purification, isolation of the individual enzymatic fractions was based exclusively on the presence of 3-HK transamination activity of the individual fractions. Surprisingly, however, the sequence data of the transaminase clone that was isolated based on the partial internal sequences of the purified HKT showed a considerable sequence homology with those of AGTs, with no apparent sequence homology to mammalian KAT (see "Results"). Obviously it was necessary to determine the identity of the enzyme. The HKT activity of the purified enzyme was assayed using 3-HK, L-kynurenine, or DL-kynurenine as amino group donors (10 mM) and pyruvate, glyoxylate, alpha -ketoglutarate, alpha -ketoadipate, alpha -keto-gamma -methiolbutyrate, or oxaloacetate as amino acceptors (10 mM) (18, 29). The possible AGT activity of the enzyme was assayed using alanine (either the L-configuration or a mixture of D- and L-configurations) as an amino group donor and glyoxylate as an amino group acceptor, respectively. The AGT activity of the enzyme was based on detection of o-phthaldialdehyde thiol (OPT)-glycine conjugate by HPLC-ED after derivatization by OPT reagent (30). The typical reaction mixture for the AGT assay consisted of 20 mM amino donor (alanine), 10 mM amino acceptor (glyoxylate), 40 µM PLP, and 0.5 µg of HKT in a total volume of 50 µl prepared using 200 mM phosphate buffer, pH 7.0. The procedure of HPLC-ED analysis was similar to the method for aspartate aminotransferase and glutamine transaminase K activity assays (31), except for the modification of chromatographic conditions (mobile phase: 50 mM potassium phosphate (pH 4.8) containing 20% acetonitrile; flow rate: 2 ml min-1; HPLC column: an Alltech C18 reversed phase column, 30 × 70 mm with 3-µm spherical particle size). The kinetic parameters of the mosquito transaminase were calculated by fitting the experimental data to the Michaelis-Menten equation using an enzyme kinetics module (SPSS Science).

Effect of pH and Temperature on the Enzyme Activity-- The effect of pH on the activity of the purified enzyme to 3-HK, kynurenine, or alanine was studied. The reaction mixture containing 0.5 µg of HKT, either 10 mM amino donors, 10 mM pyruvate (for 3-HK or kynurenine) or 20 mM amino donors, 10 mM glyoxylate (for alanine), and 40 µM PLP in a total volume of 50 µl was prepared in 200 mM phosphate buffer (pH 6, 7, or 8), borate buffer (pH 9), or carbonate buffer (pH 10). The reaction mixture was incubated at 50 °C for 5 min, and products formed in the reaction mixture were quantitated using HPLC-UV or HPLC-ED analysis. The effect of temperature on HKT activity of the purified enzyme was determined by preincubation of a mixture of amino donors (3-HK, kynurenine, or alanine) and amino acceptors (pyruvate or glyoxylate) in phosphate buffer (pH 7.0) at 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 °C for 5 min prior to the addition of HKT. The mixture was continuously incubated for 5 min, and the amounts of products in the reaction mixtures were quantitated by HPLC-UV or HPLC-ED analysis. The same reaction mixtures containing heat-inactivated HKT (100 °C for 5 min) served as controls.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HKT Purification-- The overall process for the purification of mosquito HKT involves ammonium sulfate fractionation (40-60% saturation), heat treatment, chromatographic separation using different media, and ND-PAGE. Table I summarizes the increase in specific activity and yield after each step of purification. During chromatographic separation, HKT remained on the column by the end of the ammonium sulfate gradient (1.0-0.05 M). However, the enzyme was eluted when the column was washed with buffer A in the absence of ammonium sulfate. Phenyl-Sepharose chromatography was effective for HKT separation, and 81% of total proteins were eliminated from the HKT fractions after separation. The salt concentration of the eluted HKT fractions was low enough to allow direct application onto a DEAE-Sepharose column, and the enzyme activity was eluted from the DEAE-Sepharose column between 120 and 200 mM KCl. The relatively high concentration of KCl in the HKT-active fractions did not affect HKT binding on a hydroxyapatite column equilibrated with buffer A, and the HKT activity was eluted at 240-290 mM phosphate during gradient elution. The HKT fractions from the hydroxyapatite chromatography were separated sequentially by a UNO-Q column, a second hydroxyapatite column, and second UNO-Q column; the elution profiles of HKT activity during these chromatographic separations are illustrated in Fig. 1.

                              
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Table I
HKT purification table
The activity was assayed in a reaction mixture of 50 µl containing 10 mM 3-HK, 10 mM pyruvate, 40 µM PLP, and varying amounts of protein sample prepared in 200 mM phosphate buffer, pH 7.0. The mixture was incubated for 10 min at 50 °C, and the reaction was stopped by adding an equal volume of 0.8 M formic acid. Supernatant was obtained by centrifugation of the reaction mixture at 15,000 × g for 10 min at 4 °C and analyzed by HPLC-UV at 340 nm. Protein was determined by a Bio-Rad protein assay kit using bovine serum albumin as a standard.


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  		Fig. 1.   Schematic diagram of HKT purification. Chromatograms of hydroxyapatite and UNO-Q column chromatography are shown. The marked bar shows the active fractions, and arrows indicate HKT-active absorbance peaks during chromatography.

Electrophoresis-- Gradient ND-PAGE of the concentrated HKT fraction from the second UNO-Q column chromatography, followed by Coomassie Blue staining, resulted in the detection of two major protein bands (Fig. 2, lane A). The second band was identified as HKT based on HKT activity assays of the individual gel strips. Analysis of the eluted and concentrated HKT using SDS-PAGE and Coomassie Blue staining revealed a single protein band with a molecular mass of 42,500 (Fig. 2, lane C1). A single major absorbance peak was observed when the eluted HKT-active band was chromatographed by HPLC on a Vydac C18 reversed phase column (Fig. 2B).


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  		Fig. 2.   Purification of HKT by gradient ND-PAGE and its analysis by reversed phase chromatography and SDS-PAGE, respectively. Concentrated HKT fraction from the second UNO-Q column chromatography was electrophoresed on a 5-20% native polyacrylamide gel under conditions described under "Experimental Procedures". Lane A shows the separated HKT fraction stained with Coomassie Blue. Chromatogram in B shows the relative purity of the ND-PAGE-separated HKT band during HPLC reversed phase chromatography. Lanes C1 and C2 illustrate the position of the purified HKT and molecular markers, respectively, separated by a 10% SDS-PAGE with subsequent Coomassie Blue staining. Arrows point to either the HKT band on the gel or the HKT peak in the chromatogram.

N-terminal and Internal Sequencing-- N-terminal sequencing of the HKT band blotted on a polyvinylidene difluoride membrane identified the first 19 N-terminal residues as N-MKFTPPPSSLRGPLVIPDK. Internal sequencing of the three major peptide fragments, obtained by trypsin digestion of the purified HKT and subsequent capillary HPLC separation of the tryptic digest, resulted in the identification of partial internal peptide sequences for the individual fragments (AINMATR, VLEGPADKPF, and GLEIFVK) (see Fig. 3A).


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  		Fig. 3.   HKT sequence and its comparison with AGTs. A, nucleotide sequence and deduced amino acid sequence of A. aegypti HKT. The amino acid residues that are underlined and bold are those determined by N-terminal and internal sequencing. The asterisk represents the stop codon. The bold nucleotide sequence near the end of the 3'-untranslated region is the polyadenylation signal (AATAAA). B, sequence comparison of A. aegypti HKT with AGTs of D. melanogaster, O. cuniculus, F. catus, and H. sapiens. Ae, Dm, Rb, Fe, and Hm represent AGT or putative AGT from A. aegypti, D. melanogaster (GenBankTM accession number AE003437), O. cuniculus (GenBankTM accession number M84647), F. catus (GenBankTM accession number X75923), and H. sapiens (GenBankTM accession number X53414), respectively.

Molecular Cloning and Amino Acid Sequence Similarities-- PCR amplification of first strand cDNA pool by degenerated primers amplified a 600-bp DNA fragment. Subsequent screening of an A. aegypti larval cDNA library using the 600-bp probe at high stringency led to the isolation of 10 individual clones. Three cDNA clones (with inserts > 1350 bp) were sequenced, and their sequences were identical except for the difference in the number of nucleotides at the 5'- and 3'-untranslated regions. The isolated clones bear an 1,167-bp open reading frame coding for a protein of 389 amino acids with a predicted molecular mass of 43,239 (GenBankTM accession number AF435806), consistent with the molecular mass of the purified protein (see Fig. 2, lane C). The partial N-terminal and three partial internal amino acid sequences obtained by Edman degradation correspond to the deduced amino acid residues of 1-19, 108-114, 121-130, and 302-308, respectively (Fig. 3A). BLAST analysis of the deduced HKT sequence revealed a 52% identity with a putative AGT of Drosophila melanogaster (GenBankTM accession number AE003437) and 45-46% identity with AGTs of Oryctolagus cuniculus (GenBankTM accession number M84647), Felis catus (GenBankTM accession number X75923), and Homo sapiens (GenBankTM accession number X53414). However, a BLAST search of genomic data bases did not match any known KATs. Sequence alignment of HKT with the putative AGT of Drosophila and AGTs of three mammalian species revealed several conserved regions among them (Fig. 3B). These highly conserved regions that have provided the basis to group these proteins together likely are involved in the formation of active site for the enzyme, such as PLP and/or substrate binding domain, but there has been no information regarding the specific roles these conserved regions play for any AGT.

Expression Profile during Development-- The expression profile of the HKT was evaluated by Northern blot analysis (Fig. 4). HKT mRNA (about 1.6 kb) was detected in larvae, pupae, and developing ovaries. Levels of transcript increased from 1- to 4-day-old larvae (lanes 1-4), decreased in 5- and 6-day-old larvae and newly formed pupae (lanes 5-7), and became undetectable in 12 h pupae (lane 8), but high levels of HKT transcripts were detected in developing ovaries (lane 9).


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  		Fig. 4.   Transcription profiles of the HKT gene during development. Total RNA isolation, electrophoresis, and blotting on nylon membrane were detailed under "Experimental Procedures." The blot was sequentially probed with a 600-bp [32P]dCTP-labeled HKT probe (top panel) and a 500-bp 18 S rRNA probe as a loading control (bottom panel). Lanes (top panel) show the levels of HKT transcript in 1- to 6-day-old larvae (lanes 1-6), newly formed white pupae (lane 7), 12-h black pupae (lane 8), and ovaries from adult female 24 h after emergence (lane 9).

Substrate Specificity and Kinetic Analysis-- The purified enzyme is capable of catalyzing the transamination of both kynurenine and 3-HK with pyruvate, glyoxylate, or oxaloacetate as amino group acceptors (Table II) and the transamination of alanine with glyoxylate as an amino group acceptor. Based on the calculated Km, the enzyme showed higher affinity to 3-HK than to kynurenine and alanine. The Km of the enzyme to DL-alanine (22.8 mM) is twice that of L-alanine (11.2 mM), suggesting that the D-alanine was neither a substrate nor an inhibitor. The Km of the enzyme to L-kynurenine (6.2 mM) is smaller than that of DL-kynurenine (10 mM); accordingly, the true Km of the enzyme to the L-configuration of 3-HK (which is unavailable) could be smaller than that determined using DL-forms of 3-HK. The Vmax of enzyme to 3-HK, L-kynurenine, DL-kynurenine, L-alanine, and DL-alanine are 60.6, 24, 18, 41, and 41.6 µmol/min/mg, respectively. Other kinetic parameters are shown in Table III. Among the substrates, the calculated kcat/Km is the highest for 3-HK (655 min-1mM-1), indicating that the enzyme is much more efficient in catalyzing the transamination of 3-HK than the other substrates.

                              
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Table II
HKT co-substrate specificity
Purified HKT was incubated in the presence of keto acids and 3-HK or kynurenine as described under "Experimental Procedures." Data are expressed as a percentage of the activity of that with pyruvate as the amino acceptor and are the means of two separate experiments.

                              
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Table III
HKT kinetic parameters
The activities were measured as described under "Experimental Procedures." The parameters of HKT to the 5 substrates were calculated by fitting the experimental data to the Michaelis-Menten equation using the enzyme kinetics module.

Effect of pH and Temperature on HKT Activity-- Both the pH and temperature of the reaction mixture greatly affected HKT activity to 3-HK and alanine. Among the temperature points and pH conditions tested, the enzyme showed the highest activity around 55 °C to 3-HK and 60 °C to alanine (Fig. 5A) and pH 9.0 (Fig. 5B).


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  		Fig. 5.   Effect of pH and temperature on HKT activity. The assay methods were as described under "Experimental Procedures." Panels A and B show the activities of purified HKT under different temperatures and pH, respectively. The solid lines and dashed lines represent specific activities of HKT to 3-HK and alanine, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our previous data suggest that mosquitoes have an efficient mechanism preventing the accumulation of 3-HK through conversion of the chemically reactive and potentially toxic 3-HK to chemically stable XA by an aminotransferase-mediated reaction (18), but the enzyme responsible for catalyzing the 3-HK to XA pathway is poorly understood. In this study, we have achieved the purification of mosquito HKT, isolated its cDNA from a larval cDNA library, determined its basic biochemical characteristics, and assessed its transcription profile during mosquito development. These data provide physical evidence for the presence of a transaminase that is more active to 3-HK, and the data support a previous assumption that the enzyme plays an important physiological role in preventing overaccumulation of the chemically reactive and potentially toxic 3-HK during tryptophan oxidation in mosquitoes.

Based on transaminase activity assays using crude larval proteins, we had assumed that the mosquito HKT, although more active to 3-HK than to kynurenine, likely shared considerable sequence identity and biochemical characteristics with those of mammalian KATs. KAT from mammals catalyzes the transamination of kynurenine and 3-HK and can use both pyruvate and alpha -ketoglutarate (22) as amino acceptors. Mammalian KATs have been studied extensively because these enzymes are responsible for the production of kynurenic acid, which is a nonselective blocker of excitatory receptors and plays an important physiological role in protecting the N-methyl-D-aspartate receptors of the central nervous system from being overstimulated by excitotoxin (19, 20). They also have glutamine transaminase K and aminoadipate aminotransferase activities (21-23). However, results from this study suggest that the mosquito HKT is quite different compared with mammalian KATs. For example, except for its high specific activity to 3-HK, mosquito HKT has no detectable activity with alpha -ketoglutarate or alpha -ketoadipate as amino acceptors. The most remarkable difference between mosquito HKT and KATs, however, is the dissimilarity in their primary sequences. Mosquito HKT shows 9% sequence identity with rat KAT (GenBankTM accession number Z49696), 5% sequence identity with human KAT (GenBankTM accession number XM_011753), and 4% sequence identity with our previously isolated mosquito KAT (GenBankTM accession number AF395204). The biochemical characteristics and primary sequences of this mosquito transaminase clearly distinguish it from mammalian KATs.

Surprisingly, however, the primary sequence of mosquito HKT shares fairly high sequence identity (45-46%) with mammalian AGTs, and the enzyme can also catalyze the transamination of alanine in the presence of glyoxylate, which is the definition for a protein to be classified as AGT. It has become customary to name newly isolated sequences based on their sequence identity to the known proteins whose biochemical functions have been determined. Consequently, it is reasonable to classify this mosquito transaminase as AGT. However, the high HKT activity of this mosquito transaminase raises these fundamental questions: which activity is its primary and main function in mosquitoes, and do AGTs from other sources also have the same HKT activity?

Among the available AGT and putative AGT sequences, the putative AGT of Drosophila shares the highest identity to the mosquito transaminase sequence (52%). Therefore, it was presumed that the putative Drosophila AGT would likely have the same biochemical activities as those of the mosquito enzyme. Recently, we achieved the functional expression of both the mosquito HKT and the putative Drosophila AGT using an insect/baculovirus expression system. Our preliminary data showed that the recombinant mosquito enzyme has both HKT and AGT activity, but it is more efficient catalyzing the transamination of 3-HK (kcat/Km = 789 ± 322 min-1mM-1) than that of alanine (kcat/Km = 67 ± 11 min-1mM-1) in the presence of glyoxylate, which is similar to the natural enzyme from mosquito larvae. In contrast, the Drosophila recombinant protein has a high AGT activity, but it completely lacks HKT activity.2 The high sequence identity of the mosquito transaminase with other AGTs suggests that they are evolved from the same ancestry, but it seems apparent that the major biochemical function of this mosquito enzyme has deviated considerably from that of other AGTs.

The accumulation of a high concentration of XA during larval and egg development, the detection of high HKT activity in mosquito larvae, and the high activity of the purified enzyme to 3-HK provide the basis for suggesting its major role in the transamination of 3-HK. In mosquitoes, 3-HK is used for the production of ommochromes (the major eye pigments in some insects). Compound eye development is a major physiological event in insects during pupal development, and eye pigmentation proceeds during pupal and earlier adult stages. Coincidentally, HKT activity becomes essentially undetectable in the mosquito during pupal and earlier adult stages (except during egg development after bloodfeeding), while accumulation of 3-HK and ommochrome is observed in the compound eyes (32). The down-regulation of HKT and the accumulation of 3-HK during pupal and earlier adult stages provide additional support for its role in 3-HK transamination. In addition, kinetic analysis suggests that the mosquito enzyme is more efficient in catalyzing the transamination of 3-HK to alanine, although the DL configuration of 3-HK was used for comparison.

Based on the above analysis, it is clear that the mosquito HKT plays an indispensable physiological role in tryptophan catabolism of mosquitoes. The accumulation of high concentrations of XA during larval development, the correlation between the HKT activity profile and the levels of XA in mosquitoes, the correlation between the transcriptional profile and the HKT activity profile, and the high efficiency of the purified enzyme to 3-HK provide convincing evidence for HKT's role in tryptophan catabolism in mosquitoes. Accordingly, it is apparent that the primary function of this enzyme is the transamination of 3-HK to XA. However, because the enzyme also has the typical AGT activity, it seems more appropriate to name this enzyme mosquito HKT/AGT. These results should serve as a useful reference in the study of similar enzymes from other mosquitoes. In addition, it is anticipated that these results should promote comparative studies between the mosquito HKT/AGT and mammalian AGTs.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AI 44399.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 217-244-3913; Fax: 217-244-7421; E-mail: jli2@uiuc.edu.

Published, JBC Papers in Press, March 5, 2002, DOI 10.1074/jbc.M201202200

2 Q. Han and J. Y. Li, unpublished data.

    ABBREVIATIONS

The abbreviations used are: 3-HK, 3-hydroxy-DL-kynurenine; AGT, alanine glyoxylate aminotransferase; HKT, 3-hydroxykynurenine transaminase; KAT, kynurenine aminotransferase; beta -ME, beta -mercaptoethanol; ND-PAGE, non-denaturing polyacrylamide gel electrophoresis; PLP, pyridoxal-5'-phosphate; XA, xanthurenic acid; HPLC-ED, high pressure liquid chromatography with electrochemical detection; HPLC-UV, high pressure liquid chromatography with ultraviolet detection; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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