Dihydroorotate dehydrogenases (DHOD) catalyse the oxidation of (S)-dihydroorotate to orotate in the in vivo synthesis of UMP, the precursor of pyrimidine nucleotides, building blocks of both DNA and RNA. These enzymes have a flavin mononucleotide (FMN) cofactor in common, which is involved in the transfer of a hydride ion from the substrate to the electron acceptor. According to their amino acid sequences DHOD´s can be divided into two main families [1]. Family 1 consists of cytosolic enzymes from Gram-positive bacteria, while DHOD´s from eukaryotes and Gram-negative bacteria belong to family 2 and are membrane associated. Family 2 enzymes use long-chain ubiquinones as electron acceptors, and the electrons generated are delivered to the respiratory chain. The catalytically active residue in the family 1 enzymes is a cysteine, while in the family 2 enzymes it is a serine. DHOD is the rate limiting enzyme in the de novo pyrimidine biosynthetic pathway. Human DHOD belongs to family 2 and its inhibition by quinone analogues is currently investigated as a potential means of treating immune based diseases. Until recently only structures representative of family 1 enzymes were known.

A Se-edge MAD data set was collected at the BM30A (FIP) beamline on the Se-methionine substituted form of the E. coli DHOD, belonging to family 2. The protein (37 kDa) was co-crystallised with the reaction product orotate (crystal space group: P41212, a=b=119.7 Å, c=295.97 Å). Due to the long c-axis, extreme care had to be taken to optimise the resolution and avoid spot overlaps by selecting the correct detector to crystal distance. The 2.5 Å resolution data were collected at 3 wavelengths with 120 s exposure per image (0.5° oscillation) on a MAR345 image plate detector. The data was integrated and scaled using DENZO and SCALEPACK (global Rmerge ~ 8%, Rano ~ 5%, completeness ~ 97.5%). The positions for 24 out of the 28 expected Se-sites per asymmetric unit (4 molecules/a.u.) were localised using the program SOLVE and further refined by SHARP. The first maps calculated employing NCS-averaging and DM allowed the tracing of the complete structure except for the first two residues in the N-terminus. Refinement of the structure was completed using CNS (R=17.9%, Rfree=21.9%, resolution range 20-2.5 Å). The /ß-barrel structure common to all other known DHOD structures is maintained, with the FMN group and orotate binding site situated on the top of the barrel. The structure has an extended N-terminal, which forms two helixes situated on one side of the barrel (Figure 17), which are proposed as the membrane associated part of the protein. Though the sequence identity with DHOD´s from family 1 is low, the active site and the orotate binding pattern is conserved. The substitution of the catalytic cysteine found in family 1 enzymes to a serine is confirmed, and an increase in the hydrophobic character of its environment is observed. This increase involves a substitution of a solvent oriented Glu (family 1 structures) into a Leu of which the side chain is turned inwards, shielding the active site serine.

Reference
[1] O. Björnberg, P. Rowland, S. Larsen, K.F. Jensen, Biochemistry 36, 16197-16205 (1997).

Authors
S. Nørager (a), O. Björnberg (b), K.F. Jensen (b), S. Larsen (a).

(a) Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen (Denmark)
(b) Institute for Molecular Biology, University of Copenhagen (Denmark)