Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane‐associated N‐terminal domain

• Published in: Protein science 2004-04, Vol.13 (4), p.1031-1042
• Main Authors: Hansen, Majbritt, Le Nours, Jérôme, Johansson, Eva, Antal, Torben, Ullrich, Alexandra, Löffler, Monika, Larsen, Sine
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Aniline Compounds - chemistry; Aniline Compounds - metabolism; Animals; Atovaquone; Biphenyl Compounds - chemistry; Biphenyl Compounds - metabolism; brequinar; Catalysis; Crystallography, X-Ray; DCIP, 2,3‐dichlorophenolindophenol; DHO, dihydroorotate; DHOD, dihydroorotate dehydrogenase; DHODA, Lactococcus lactis DHOD A; DHODB, Lactococcus lactis DHOD B; DHODC, Escherichia coli DHOD; DHODH, human DHOD; DHODH‐breq, structure of human DHOD in complex with brequinar; DHODH‐lefl, structure of human DHOD structure in complex with leflunomide; DHODR, rat DHOD; DHODR‐ato, structure of rat DHOD structure in complex with atovaquone; DHODR‐breq, structure of rat DHOD in complex with brequinar; dihydroorotate dehydrogenase; domain movement; Drug Design; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; HEPES, 4–(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid; Hydrogen Bonding; Hydroxybutyrates - chemistry; Hydroxybutyrates - metabolism; Immunosuppressive Agents - chemistry; Immunosuppressive Agents - metabolism; inhibitor binding; membrane association; Models, Molecular; Molecular Sequence Data; Molecular Structure; MPD, 2‐methyl 2,4‐pentanediol; Naphthoquinones - chemistry; Naphthoquinones - metabolism; Orotic Acid - analogs & derivatives; Orotic Acid - chemistry; Oxidoreductases Acting on CH-CH Group Donors - chemistry; Oxidoreductases Acting on CH-CH Group Donors - metabolism; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Q0, 2,3‐dimethoxy‐5‐methyl‐benzoquinone; Rats; rmsd, root‐mean‐square deviation; Sequence Alignment; Substrate Specificity; TRIS, Tris‐(hydroxymethyl)aminomethane, 2‐amino‐2–(hydroxymethyl)‐1,3‐propanediol
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.03533004

The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti‐proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N‐terminal extension, which folds into a separate domain comprising two α‐helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N‐terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 Å movement of the first residue in the first α‐helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane‐bound rat DHOD and membrane‐associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.