title: Purification and biochemical characterization of a recombinant Anopheles gambiae tryptophan 2,3-dioxygenase expressed in Escherichia coli.
abstract: In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway. TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring. Despite the central role exerted by kynurenines in the physiology of living organisms, only a few insect TDOs have been subjected to biochemical characterization in vitro. We performed a RT-PCR-based analysis of the tissue distribution of TDO mRNA in A. gambiae that revealed a ubiquitous expression of the gene, thus further underlining the importance of the enzyme in the mosquito biology. We developed an expression/purification procedure yielding pure and active recombinant A. gambiae TDO. Spectral analyses showed that the enzyme was purified in its heme-ferric form that was subsequently used to determining the Michaelis-Menten constants of the TDO catalyzed reaction in the presence of reducing agents. The screening of a number of compounds as potential TDO modulators showed that several kynurenines and other Tryptophan-derived molecules interfere with the enzyme activity in vitro. Our study could contribute to understanding TDO regulation in vivo and to the identification of inhibitors to be used to alter Tryptophan homeostasis in the malaria vector.
authors.affiliation: DiSCAFF, University of Piemonte Orientale "A. Avogadro", Via Bovio, 6, 28100 Novara, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Paglino
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Lombardo
authors.forename: Bruno
authors.initials: B
authors.lastname: Arcà
authors.forename: Menico
authors.initials: M
authors.lastname: Rizzi
authors.forename: Franca
authors.initials: F
authors.lastname: Rossi
mesh_terms: D000818:Animals; D000852:Anopheles / Q000201:enzymology*; D004926:Escherichia coli / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D014366:Tryptophan Oxygenase / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014366:Tryptophan Oxygenase