THE CRYSTAL-STRUCTURE OF THE FLAVIN-CONTAINING ENZYME DIHYDROOROTATE DEHYDROGENASE-A FROM LACTOCOCCUS-LACTIS

Background: Dihydroorotate dehydrogenase (DHOD) is a flavin mononucleo tide containing enzyme, which catalyzes the oxidation of (S)-dihydroor otate to orotate, the fourth step in the de novo biosynthesis of pyrim idine nucleotides. Lactococcus lactis contains two genes encoding diff erent functional DHODs whose sequences are only 30% identical. One of these enzymes, DHODA, is a highly efficient dimer, while the other, DH ODB, shows optimal activity only in the presence of an iron-sulphur cl uster containing protein with which it forms a complex tetramer. Seque nce alignments have identified three different families among the DHOD s: the two L. lactis enzymes belong to two of the families, whereas th e enzyme from E. coli is a representative of the third. As no three-di mensional structures of DHODs are currently available, we set out to d etermine the crystal structure of DHODA from L. lactis. The difference s between the two L. lactis enzymes make them particularly interesting for studying flavoprotein redox reactions and for identifying the dif ferences between the enzyme families. Results: The crystal structure o f DHODA has been determined to 2.0 Angstrom resolution. The enzyme is a dimer of two crystallographically independent molecules related by a non-crystallographic twofold axis. The protein folds into an alpha/be ta barrel with the flavin molecule sitting between the top of the barr el and a subdomain formed by several barrel inserts. Above the Ravin i soalloxazine ring there is a small water filled cavity, completely bur ied beneath the protein surface and surrounded by many conserved resid ues. This cavity is proposed as the substrate-binding site. Conclusion s: The crystal structure has allowed the function of many of the conse rved residues in DHODs to be identified: many of these are associated with binding the flavin group. Important differences were identified i n some of the active-site residues which vary across the distinct DHOD families, implying significant mechanistic differences. The substrate cavity, although buried, is located beneath a highly conserved loop w hich is much less ordered than the rest of the protein and may be impo rtant in giving access to the cavity. The location of the conserved re sidues surrounding this cavity suggests the potential orientation of t he substrate.