Protein Science, Vol 7, Issue 6 1269-1279, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

The crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function

P. ROWLAND, O. BJORNBERG, F. S. NIELSEN, K. F. JENSEN and S. LARSEN
Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen O, Denmark

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved 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 hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.
CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. G. Couto, M. C. Nonato, and A. J. Costa-Filho Defects in Vesicle Core Induced by Escherichia coli Dihydroorotate Dehydrogenase Biophys. J., March 1, 2008; 94(5): 1746 - 1753. [Abstract] [Full Text] [PDF]

				J. P. Combe, J. Basran, P. Hothi, D. Leys, S. E. J. Rigby, A. W. Munro, and N. S. Scrutton Lys-D48 Is Required for Charge Stabilization, Rapid Flavin Reduction, and Internal Electron Transfer in the Catalytic Cycle of Dihydroorotate Dehydrogenase B of Lactococcus lactis J. Biol. Chem., June 30, 2006; 281(26): 17977 - 17988. [Abstract] [Full Text] [PDF]

				R. Iyer, N. S. Baliga, and A. Camilli Catabolite Control Protein A (CcpA) Contributes to Virulence and Regulation of Sugar Metabolism in Streptococcus pneumoniae J. Bacteriol., December 15, 2005; 187(24): 8340 - 8349. [Abstract] [Full Text] [PDF]

				K. Wang and R. Samudrala FSSA: a novel method for identifying functional signatures from structural alignments Bioinformatics, July 1, 2005; 21(13): 2969 - 2977. [Abstract] [Full Text] [PDF]

				M. Hansen, J. Le Nours, E. Johansson, T. Antal, A. Ullrich, M. Loffler, and S. Larsen Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain Protein Sci., April 1, 2004; 13(4): 1031 - 1042. [Abstract] [Full Text] [PDF]

				S. Norager, S. Arent, O. Bjornberg, M. Ottosen, L. L. Leggio, K. F. Jensen, and S. Larsen Lactococcus lactis Dihydroorotate Dehydrogenase A Mutants Reveal Important Facets of the Enzymatic Function J. Biol. Chem., August 1, 2003; 278(31): 28812 - 28822. [Abstract] [Full Text] [PDF]

				M. B. Ottosen, O. Bjornberg, S. Norager, S. Larsen, B. A. Palfey, and K. F. Jensen The dimeric dihydroorotate dehydrogenase A from Lactococcus lactis dissociates reversibly into inactive monomers Protein Sci., November 1, 2002; 11(11): 2575 - 2583. [Abstract] [Full Text] [PDF]

				D. Dobritzsch, S. Ricagno, G. Schneider, K. D. Schnackerz, and Y. Lindqvist Crystal Structure of the Productive Ternary Complex of Dihydropyrimidine Dehydrogenase with NADPH and 5-Iodouracil. IMPLICATIONS FOR MECHANISM OF INHIBITION AND ELECTRON TRANSFER J. Biol. Chem., April 5, 2002; 277(15): 13155 - 13166. [Abstract] [Full Text] [PDF]

				G. Pujadas and J. Palau Molecular mimicry of substrate oxygen atoms by water molecules in the {beta}-amylase active site Protein Sci., August 1, 2001; 10(8): 1645 - 1657. [Abstract] [Full Text] [PDF]

				Z. Gojkovic, M. P. B. Sandrini, and J. Piskur Eukaryotic {beta}-Alanine Synthases Are Functionally Related but Have a High Degree of Structural Diversity Genetics, July 1, 2001; 158(3): 999 - 1011. [Abstract] [Full Text] [PDF]

				G. Gadda, A. Banerjee, L. J. Dangott, and P. F. Fitzpatrick Identification of a Cysteine Residue in the Active Site of Nitroalkane Oxidase by Modification with N-Ethylmaleimide J. Biol. Chem., October 6, 2000; 275(41): 31891 - 31895. [Abstract] [Full Text] [PDF]