THE CRYSTAL-STRUCTURE OF LACTOCOCCUS-LACTIS DIHYDROOROTATE DEHYDROGENASE-A COMPLEXED WITH THE ENZYME REACTION-PRODUCT THROWS LIGHT ON ITS ENZYMATIC FUNCTION

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-di hydroorotate to orotate, the fourth step and only redox reaction in th e de novo biosynthesis of pyrimidine nucleotides. A description is giv en of the crystal structure of Lactococcus lactis dihydroorotate dehyd rogenase A (DHODA) complexed with the product of the enzyme reaction o rorate, The structure of the complex to 2.0 Angstrom, resolution has b een compared with the structure of the native enzyme. Th active site o f DHODA is known to contain a water filled cavity buried beneath a hig hly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA fl avin isoalloxazine ring, causing only small movements of the surroundi ng protein residues. The orotate is completely buried beneath the prot ein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four co nserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193) , the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hy drogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotat e are considered using the orotate complex as a basis for molecular mo deling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the diffe rent families of DHODs is reviewed, along with implications for differ ences in substrate binding and in the catalytic mechanisms between the se families.