title: The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis.
abstract: Leishmania major dihydro-orotate dehydrogenase (DHODHLm) has been considered as a potential therapeutic target against leishmaniasis. DHODHLm, a member of class 1A DHODH, oxidizes dihydro-orotate (DHO) to orotate (ORO) during pyrimidine biosynthesis using fumarate (FUM) as the oxidizing substrate. In the present study, the chemistry of reduction and reoxidation of the flavin mononucleotide (FMN) cofactor in DHODHLm was examined by steady- and pre-steady state kinetics under both aerobic and anaerobic environments. Our results provide for the first time the experimental evidence of co-operative behaviour in class 1A DHODH regulated by DHO binding and reveal that the initial reductive flavin half-reaction follows a mechanism with two steps. The first step is consistent with FMN reduction and shows a hyperbolic dependence on the DHO concentration with a limiting rate (kred) of 110±6 s(-1) and a K(DHO) d of 180±27 μM. Dissociation of the reduced flavin-ORO complex corresponds to the second step, with a limiting rate of 6 s(-1). In the oxidative half-reaction, the oxygen-sensitive reoxidation of the reduced FMN cofactor of DHODHLm by FUM exhibited a hyperbolic saturation profile dependent on FUM concentration allowing estimation of K(FUM) d and the limiting rate (kreox) of 258±53 μM and 35±2 s(-1), respectively. Comparison between steady- and pre-steady-state parameters together with studies of interaction for DHODHLm with both ORO and succinate (SUC), suggests that ORO release is the rate-limiting step in overall catalysis. Our results provide evidence of mechanistic differences between class 1A and class 2 individual half-reactions to be exploited for the development of selective inhibitors.
authors.affiliation: Laboratório de Cristalografia de Proteínas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903, Ribeirão Preto, Brazil.
authors.forename: Renata A G
authors.initials: RA
authors.lastname: Reis
authors.affiliation: Department of Biochemistry and Molecular and Cellular Biology, and Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, E-50009, Zaragoza, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Ferreira
authors.affiliation: Department of Biochemistry and Molecular and Cellular Biology, and Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, E-50009, Zaragoza, Spain.
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
authors.affiliation: Laboratório de Cristalografia de Proteínas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903, Ribeirão Preto, Brazil cristy@fcfrp.usp.br.
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Nonato
mesh_terms: D055162:Biocatalysis; D004138:Dinitrocresols / Q000737:chemistry; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D009963:Orotic Acid / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019802:Succinic Acid / Q000737:chemistry
chemical_list: D004138:Dinitrocresols; D011994:Recombinant Proteins; C024132:4,6-dinitro-o-cresol; D009963:Orotic Acid; D019802:Succinic Acid; D010088:Oxidoreductases; C019999:dehydroorotate dehydrogenase
keywords: Leishmania major; co-operativity; dihydro-orotate dehydrogenase; enzyme mechanism; flavoprotein; nucleoside/nucleotide biosynthesis