9VPZ

Crystal structure of wild-type Trypanosoma brucei DHODH in FMN-reduced, ligand-free form

Summary for 9VPZ

• Entry DOI: 10.2210/pdb9vpz/pdb
• Descriptor: Dihydroorotate dehydrogenase (fumarate), 1-DEOXY-1-(7,8-DIMETHYL-2,4-DIOXO-3,4-DIHYDRO-2H-BENZO[G]PTERIDIN-1-ID-10(5H)-YL)-5-O-PHOSPHONATO-D-RIBITOL, MALONATE ION, ... (6 entities in total)
• Functional Keywords: ping-pong reaction mechanism, substrate/product-enzyme interactions., flavoprotein
• Biological source: Trypanosoma brucei brucei TREU927
• Total number of polymer chains: 4
• Total formula weight: 141427.08

Authors

Kubota, T.,Tani, O.,Yamasaki, K. (deposition date: 2025-07-04, release date: 2025-11-26, Last modification date: 2025-12-10)

Primary citation

Tani, O.,Kubota, T.,Yamasaki, T.,Hirokawa, T.,Furukawa, K.,Yamasaki, K.
Structural Basis of Redox-Dependent Affinities of Dihydroorotate Dehydrogenase for Its Substrates and Products.
J.Mol.Biol., 438:169544-169544, 2025

PubMed Abstract: Dihydroorotate dehydrogenase (DHODH), containing a cofactor, flavin mononucleotide (FMN), catalyzes the oxidation of dihydroorotate to orotate. In Trypanosoma brucei, the causative parasite of African trypanosomiasis, the DHODH enzyme (TbDHODH) couples this reaction with the reduction of fumarate to succinate. These reactions occur successively in the same catalytic site through a ping-pong bi-bi mechanism. FMN is reduced in the first step and re-oxidized in the second one. Here, we investigated how the redox state of FMN influences the binding of substrates and products to TbDHODH by NMR analyses of a catalytically impaired mutant. The affinities for the substrates, dihydroorotate and fumarate, were redox-dependent: dihydroorotate preferred the oxidized state, whereas fumarate preferred the reduced state. One of the products, succinate, exhibited no detectable binding to either of the redox states, suggesting its efficient product release. In contrast, the product of the first-half reaction, orotate, showed stronger binding to the oxidized state, suggesting that product inhibition may occur unless orotate is rapidly consumed in the subsequent biosynthetic step. The structural basis of these findings was elucidated by crystallography of the complexes with the above ligands in both redox states, along with quantitative analyses of the interactions using quantum chemical calculations. Upon reduction, the FMN isoalloxazine ring gains two additional hydrogens and becomes more bent at its center. These changes perturb the interactions among the ligands, flavin, and surrounding residues (notably Lys44, Asn68, and Asn195), primarily in the electrostatic term, which quantitatively explains the difference in binding affinities.

PubMed: 41241202
DOI: 10.1016/j.jmb.2025.169544

Experimental method

X-RAY DIFFRACTION (1.75 Å)