The structures of human dihydroorotate dehydrogenase with and without inhibitor reveal conformational flexibility in the inhibitor and substrate binding sites

Biochemistry. 2008 Aug 26;47(34):8929-36. doi: 10.1021/bi8003318. Epub 2008 Aug 2.

Abstract

Inhibitors of dihydroorotate dehydrogenase (DHODH) have been suggested for the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections. Here we present the structures of N-terminally truncated (residues Met30-Arg396) DHODH in complex with two inhibitors: a brequinar analogue (6) and a novel inhibitor (a fenamic acid derivative) (7), as well as the first structure of the enzyme to be characterized without any bound inhibitor. It is shown that 7 uses the "standard" brequinar binding mode and, in addition, interacts with Tyr356, a residue conserved in most class 2 DHODH proteins. Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone undergo changes in conformation upon inhibitor binding. Using our data, the loop regions of residues Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/cofactor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites / genetics
  • Biphenyl Compounds / chemistry
  • Biphenyl Compounds / metabolism
  • Carboxylic Acids / chemistry
  • Carboxylic Acids / metabolism
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / metabolism*
  • Flavin Mononucleotide / metabolism
  • Humans
  • Naphthalenes / chemistry
  • Naphthalenes / metabolism
  • Orotic Acid / analogs & derivatives
  • Orotic Acid / chemistry
  • Orotic Acid / metabolism
  • Oxidoreductases Acting on CH-CH Group Donors / chemistry
  • Oxidoreductases Acting on CH-CH Group Donors / genetics
  • Oxidoreductases Acting on CH-CH Group Donors / metabolism*
  • Protein Binding
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Ubiquinone / metabolism
  • ortho-Aminobenzoates / chemical synthesis
  • ortho-Aminobenzoates / chemistry
  • ortho-Aminobenzoates / metabolism

Substances

  • Biphenyl Compounds
  • Carboxylic Acids
  • Enzyme Inhibitors
  • Naphthalenes
  • Recombinant Proteins
  • ortho-Aminobenzoates
  • Ubiquinone
  • 4,5-dihydroorotic acid
  • 1-naphthoic acid
  • brequinar
  • Orotic Acid
  • Flavin Mononucleotide
  • fenamic acid
  • Oxidoreductases Acting on CH-CH Group Donors
  • dihydroorotate dehydrogenase