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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis

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Reis, Renata A. G. [1] ; Ferreira, Patricia [2, 3] ; Medina, Milagros [2, 3] ; Nonato, M. Cristina [1]
Total Authors: 4
[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Lab Cristal Prot, BR-14040903 Ribeirao Preto - Brazil
[2] Univ Zaragoza, Dept Biochem & Mol & Cellular Biol, E-50009 Zaragoza - Spain
[3] Univ Zaragoza, Inst Biocomputat & Phys Complex Syst BIFI, E-50009 Zaragoza - Spain
Total Affiliations: 3
Document type: Journal article
Source: Biochemical Journal; v. 473, n. 5, p. 651-660, MAR 1 2016.
Web of Science Citations: 5

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 (k(red)) of 110 +/- 6 s(-1) and a K-d(DHO) of 180 +/- 27 mu M. 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-d(FUM) and the limiting rate (k(reox)) of 258 +/- 53 mu M 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. (AU)

FAPESP's process: 11/23504-9 - Exploring structure-function relationship of DHODH enzyme in the development of new molecules with trypanocidal and leishmanicidal activities
Grantee:Renata Almeida Garcia Reis
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 12/25075-0 - Development of leishmanicidal drugs based on the selective inhibition of the enzyme dihydroorotate dehydrogenase
Grantee:Maria Cristina Nonato
Support type: Regular Research Grants