Progress 06/01/99 to 05/01/08
Outputs
This project studies tryptophan metabolism in mosquitoes. Emphasis of the project is placed on the biochemical characterization of mosquito kynurenine aminotransferase and 3-hydroxykynurenine transaminase. In mosquitoes, most of the dietary tryptophan is oxidized to kynurenine and then hydroxylated to 3-hydroxykynurenine (3-HK). 3-HK oxidizes easily in the presence of oxygen, promoting the production of reactive oxygen species. Our data suggest transamination of the chemically reactive and potentially toxic 3-HK to XA by mosquito 3-HK transaminase (HKT) is the mechanism used by the mosquitoes to detoxifying 3-HK. Our recent studies indicate that the kynurenine to kynurenic acid pathway mediated by a kunurenine aminotransferase (KAT) also plays important physiological role in mosquito central nervous system. Comparative studies revealed some major differences in biochemical characteristics between the mosquito enzyme and the counterpart enzyme from humans. Strucutral
characterizations of human and mosquito kynurenine aminotransferases have been accomplished and structural characterization of the mosquito HKT is underway.
Impacts
Transamination of 3-hydroxykynurenine to xanthurenic acid is a critical pathway for mosquitoes and the protein involved in the pathway is a potential target for mosquito control.
Publications
- Beerntsen B.T. and Li J. (2005) Plasmodium Development in White-eye (khw) and Transformed Strains (kh43) of Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology (in press).
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Progress 10/01/03 to 09/30/04
Outputs
This project studies tryptophan metabolism in mosquitoes. Emphasis of the project is placed on the biochemical characterization of mosquito kynurenine aminotransferase and 3-hydroxykynurenine transaminase. In mosquitoes, most of the dietary tryptophan is oxidized to kynurenine and then hydroxylated to 3-hydroxykynurenine (3-HK). 3-HK oxidizes easily in the presence of oxygen, promoting the production of reactive oxygen species. Our data suggest transamination of the chemically reactive and potentially toxic 3-HK to XA is the mechanism used by the mosquitoes to detoxifying 3-HK. Our recent studies indicate that the kynurenine to kynurenic acid pathway also plays important physiological role in mosquito central nervous system. Comparative studies revealed some major differences in biochemical characteristics between the mosquito enzyme and the counterpart enzyme from humans. Structural characterization of the mosquito KAT and HKT is underway.
Impacts
Transamination of 3-hydroxykynurenine to xanthurenic acid is a critical pathway for mosquitoes and the protein involved in the pathway is a potential target for mosquito control.Han Q., Li J.S. and Li J. (2004) pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I. European Journal of Biochemistry. 271: 4804-4814.
Publications
- Han Q., Li J.S. and Li J. (2004) pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I. European Journal of Biochemistry. 271: 4804-4814.
- Han Q. and Li J. (2004) Cysteine and keto acids modulate mosquito kynurenine aminotransferase catalyzed kynurenic acid production. FEBS letter 557: 381-385.
- Rossi F., Han, Li J. and Rizzi M. (2004) Structure of human kynurenine aminotransferase I. A key enzyme for the synthesis of the potent neuroactive compound kynurenic acid. Journal of Biological Chemistry 279: 50214-50220.
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Progress 10/01/02 to 09/30/03
Outputs
This project studies the tryptophan metabolism in mosquitoes. Emphasis of the project is placed on the structure/function relationship of enzymes involved in the tryptophan oxidation pathway, which includes tryptophan dioxygenase, kynurenine hydroxylase, kynurenine transaminase and 3-hydroxykynurenine transaminase. In mosquitoes, most of the dietary tryptophan is oxidized to kynurenine and then hydroxylated to 3-hydroxykynurenine (3-HK). However, 3-HK oxidizes easily in the presence of oxygen, promoting the production of reactive oxygen species. Our data suggest transamination of the chemically reactive and potentially toxic 3-HK to XA is the mechanism used by the mosquitoes to detoxifying 3-HK. Our studies dealing with the tryptophan to XA pathway resulted in the cloning and biochemical characterization of the major enzymes involved in the pathway. Currently, we are working on the detailed biochemical and structural characterization of kynurenine transaminase and
3-HK transaminase.
Impacts
Transamination of 3-hydroxykynurenine to xanthurenic acid is a critical pathway for mosquitoes and the proteins involved in the pathway is a potential target for mosquito control.
Publications
- Han Q., Calyo E., Marinotti O., Fang J., Rizzi M., James A.A. and Li J. (2003) Analysis of the wild-type and mutant genes encoding the enzyme kynurenine monooxygenase of the yellow fever mosquito, Aedes aegypti. Insect Molecular Biology 12: 483-90.
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Progress 10/01/01 to 09/30/02
Outputs
This project studies the tryptophan metabolism in mosquitoes. Emphasis of the project is placed on the biochemical characterization of an enzyme involved in catalyzing the transamination of 3-hydroxykynurenine to xanthurenic acid in the mosquitoes. In mosquitoes, most of the dietary tryptophan is oxidized to kynurenine and then hydroxylated to 3-hydroxykynurenine (3-HK). However, 3-HK oxidizes easily in the presence of oxygen, promoting the production of reactive oxygen species. Our data suggest transamination of the chemically reactive and potentially toxic 3-HK to XA is the mechanism used by the mosquitoes to detoxifying 3-HK. Our studies dealing with the tryptophan to XA pathway resulted in the cloning and biochemical characterization of the major enzymes involved in the pathway. Currently, we are working on the detailed biochemical and structural characterization of kynurenine aminotransferase and 3-HK transaminase.
Impacts
Transamination of 3-hydroxykynurenine to xanthurenic acid is a critical pathway for mosquitoes and the protein involved in the pathway is a potential target for mosquito
Publications
- Han Q. and Li J. (2002) Comparative study of Aedes aegypti 3-hydroxykynurenine transaminase/alanine glyoxylate transaminase and Drosophila alanine glyoxylate transaminase. FEBS Letters 527:199-204.
- han Q., Fang J. and Li J. (2002) 3-hydroxykynurenine transaminase, identity with alanine glyxoxylate transaminase, a probable detoxification protein in Aedes aegypti. Journal of Biological Chemistry 277: 15781-15787.
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Progress 10/01/00 to 09/30/01
Outputs
This project studies the biochemistry of tryptophan metabolism in mosquitoes with emphasis on the biochemical and molecular regulation of 3-hydroxykynurenine transaminase (HKT) during mosquito development. Oxidation of tryptophan to 3-hydroxykynurenine (3-HK) is a major pathway of tryptophan metabolism in Aedes aegypti mosquitoes. However, 3-HK is oxidized easily under physiological conditions, which stimulates the production of reactive oxygen species. Our data suggest that mosquitoes control the level of 3-HK by its transamination to chemical stable xanthurenic acid (XA). Three enzymes, namely tryptophan (TRP) oxygenase, kynurenine monooxygenase (KMO) and 3-HK transaminase (HKT), are involved in the pathway from tryptophan to XA. During the past year, we have focused on characterizing these three enzymes. Isolation and functional expression of A. aegypti KMO and TRP oxygenase. Using degenerate primers based on the conserved sequence data for KMO and tryptophan
oxygenase in the databases, we amplified partial cDNA for both A. aegypti KMO and tryptophan oxygenase and isolated full-length cDNA clones for both enzymes. Expression of both recombinant KMO baculovirus and TRP baculovirus in Sf9 insect cells resulted in successful expression of active enzymes and biochemical characterization of both recombinant enzymes is underway. Isolation and characterization of A. aegypti kynurenine aminotransferase (KAT). In mammals, there is a kynurenine aminotransferase (KAT) capable of catalyzing the transamination of both kynurenine and 3-HK. It was presumed that the mosquito transaminase, although more active to 3-HK, was related to the mammalian KATs and that they might share a considerable sequence homology. We designed degenerate primers based on the conserved regions of the mammalian KATs, amplified a cDNA fragment from a mosquito larval cDNA pool, and then isolated a putative KAT clone. The expressed protein unexpectedly showed no 3-HK transamination
activity. These data demonstrate that A. aegypti has a transaminase that is more specific for kynurenine. Purification and functional characterization of A. aegypti HKT. Inability of the mosquito KAT to catalyze the transamination of 3-HK suggests the presence of a difference transaminase for 3-HK transamination in A. aegypti. Using various separation techniques, we purified the mosquito HKT from A. aegypti larvae and determined that the enzyme is highly active to 3HK. Cloning of TRP oxygenase, KMO, KAT and HKT and their functional expression provide the necessary basis towards completely understanding the biochemical characteristics and molecular regulation of these enzymes involved in tryptophan catabolism in mosquitoes.
Impacts
(N/A)
Publications
- Han Q. and Li J. (2001) Functional characterization of 3-hydroxykynurenine aminotransferase from Aedes aegypti mosquitoes (in manuscript).
- Fang J. and Li J. (2001) Isolation, characterization, and functional expression of kynurenine aminotransferase cDNA from the yellow fever mosquito, Aedes aegypti. Insect Biochemistry and Molecular Biology (in press).
- Han Q., Fang J.M. and Li J. (2001) Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity to aspartate aminotransferase. Bichemical Journal 360: 617-623.
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Progress 10/01/99 to 09/30/00
Outputs
This project studies the biochemistry of tryptophan metabolism in mosquitoes with emphasis on the biochemical and molecular regulation of 3-hydroxykynurenine transaminase (HKT) during mosquito development. During the past year, we have been working on constructing an Aedes aegypti larval cDNA library, isolating HKT gene, and purifying HKT for its biochemical characterization. (1) cDNA library construction. To isolate A. aegypti HKT gene, it is necessary to have a quality cDNA library. We have constructed an A. aegypti larval cDNA library using a library construction kit from Stratagene (La Jolla, CA) using mRNAs isolated from 1- to 4-day old larvae. The size of the larval cDNA library is approximately 500,000 plaque forming units (pfu), which indicates a very good presentation of the mosquito genome. Restriction enzyme digestion of 34 random clones revealed that average length of inserts is 2.05 kb, and all of the random clones (34 total) carry inserts. (2) Toward
isolation of HKT gene. No sequence data for insect kynurenine transaminase or HKT are available, but the mosquito HKT is functionally related to mammalian kynurenine aminotransferases. Using degenerate primers based on sequence data from mammalian kynurenine aminotransferases, we have isolated a cDNA clone from our newly constructed larval cDNA library. Its deduced amino acid sequence shares about 40% sequence homology with those reported for mammalian kynurenine aminotransferases and contains the pyridoxal phosphate binding domain, a functional domain for transaminases. To characterize its product, the putative transaminase clone is currently being expressed using an insect-baculovirus protein expression system. (3). Purification and characterization of A. aegypti HKT. During the past year, we also achieved purification of the A. aegypti HKT directly from mosquito larvae by various chromatographic techniques plus non-denaturing electrophoresis. The isolated enzyme has a relative
molecular mass of 80,000 and 40,000 by gradient native PAGE and SDS-PAGE analysis, respectively, which indicates that the native enzyme is a homodimer. The purified enzyme is active to 3-hydroxykynurenine and kynurenine and shows a maximum activity at temperature around 55 and at pH around 6.0. More HKT is being purified for N-terminal and internal sequencing, and the sequence data will be used to compare with those of the putative transaminase clone isolated from a larval cDNA library.
Impacts
(N/A)
Publications
- Han Q., Runlin Ma and J. Li (2001) Purification and characterization of 3-hydroxykynurenine transaminase from Aedes aegypti (submitted).
- Fang J.M. and Li J. (2001) Cloning and functional expression of Aedes aegypti kynurenine/3-hydroxykynurenine aminotransferase (in preparation).
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