Structures of Trypanosoma cruzi Dihydroorotate Dehydrogenase Complexed with Substrates and Products: Atomic Resolution Insights into Mechanisms of Dihydroorotate Oxidation and Fumarate Reduction

Abstract

Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-reactions. These ligands bind to the same active site of TcDHOD, which is consistent with the one-site ping-pong Bi-Bi mechanism demonstrated by kinetic studies for family 1A DHODs. The binding of ligands to TcDHOD does not cause any significant structural changes to TcDHOD, and both reduced and oxidized FMN cofactors are in planar conformation, which indicates that the reduction of the FMN cofactor with dihydroorotate produces anionic reduced FMN. Therefore, they should be good models for the enzymatic reaction pathway of TcDHOD, although orotate and fumarate bind to TcDHOD with the oxidized FMN and dihydroorotate with the reduced FMN in the structures determined here. Cys130, which was identified as the active site base for family 1A DHOD (Fagan, R. L., Jensen, K. F., Bjornberg, O., and Palfey, B. A. (2007) Biochemistry 46, 4028-4036.), is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation represented as C 2 (delta-) and C 3 (delta+). Because of this partial charge separation, the thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that C 2 (delta-) accepts a proton from Cys130 and C 3 (delta+) a hydride (or a hydride equivalent) from reduced FMN N 5 in TcDHOD.

A QM/MM Free Energy Study of the Oxidation Mechanism of Dihydroorotate Dehydrogenase (Class 1A) from Lactococcus lactis

“…68 However, in previous theoretical results there is no hydrogen bond formed between the carboxylate group and Cys130 amino acid. 19 Then in accordance with Inaoka's publication, 24 we kept the thiol group of Cys130 deprotonated.…”

“…24 Fagan proposed that the conversion of DHO to ORO on flavin reduction in anaerobic experiments occurs by concerted mechanism. 13 Recently, austin model 1/molecular mechanics (AM1/MM) simulations were used to study the mechanism of the halfreactions in the DHOD (class 1A) from T. cruzi.…”

The Open Form Inducer Approach for Structure-Based Drug Design

“…T . cruzi DHODH (TcDHODH) belongs to family 1A and by using structures complexed with substrates and products, we previously showed that the enzyme utilizes the same site for the first-half (dihydroorotate oxidation) and second-half (fumarate reduction) reactions[16], while family 1B[17] and 2[18] utilize distinct sites for these reactions. However, there are no reports of a potent, specific inhibitors targeting family 1A DHODHs to date.…”

Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematodeAscaris suumwith the specific inhibitor flutolanil

“…The parasitic protozoan Trypanosoma cruzi utilizes bacterial-type dihydroorotate dehydrogenase (TcDHOD), which is the fourth enzyme in the pyrimidine-biosynthesis pathway and catalyzes the oxidation of dihydroorotate and the reduction of fumarate to succinate. We have elucidated the catalytic mechanisms of these sequential reactions by determination of the three-dimensional structures of TcDHOD in the ligand-free form and in complex with the substrates (dihydroorotate and fumarate), product (orotate and succinate) and inhibitor (oxonate) at atomic resolution (Inaoka et al, 2008).…”