School of Molecular and Microbial Biosciences, University of Sydney^* Division of Biomolecular Sciences, Imperial College, London, SW7 2ZA, UK^**
○Mihwa Lee^* Megan J. Maher^** Richard I. Christopherson^* J. Mitchell Guss^*

Dihydroorotase (DHOase) is a zinc metalloenzyme that catalyses the reversible cyclization of N-carbamyl-L-aspartate (CA-asp) to L-dihydroorotate (DHO) in the de novo pyrimidine biosynthetic pathway. Two different conformations of a surface loop (residues 105-115) are found in the dimeric E. coli DHOase crystallized in the presence of L-DHO [1-2]. The loop asymmetry mirrors that of the active site contents of the two subunits: DHO is bound in the active site of one subunit and CA-asp in the other active site. In the CA-asp-bound subunit, the surface loop reaches in towards the active site and makes hydrogen bonds with the bound substrate, whereas the loop forms part of the surface of the protein in the DHO-bound subunit.
To investigate the relationship between the structural states of the loop and the catalytic mechanism, 8 mutant forms of DHOase including deletion of the entire loop were generated and characterized structurally and kinetically. In the presence of L-DHO, some of these mutants first formed tetragonal crystals with one monomer in the asymmetric unit with the loop in the "out" conformation. In contrast, the wild type enzyme formed orthorhombic crystals with a dimer in the asymmetric unit with one loop in and one out. The tetragonal crystals were extremely unstable and disintegrated shortly after formation, followed by the growth of orthorhombic crystals from the remnants of the tetragonal crystals. This transformation has been explored by using a product analogue instead of L-DHO in the crystallization. The close relationship between the catalytic state of DHOase and the importance of the loop movement will be discussed based on the kinetic and structural analyses.

1. Thoden, et al. (2001) Biochem, 40, 6989
2. Lee, et al. (2005) J Mol Biol, 348, 523