Dihydroorotase (DHOase; EC 126.96.36.199) is a zinc metalloenzyme that catalyzes the reversible cyclization of N
-CA-asp) to l
-DHO) in the third step of the de novo
pyrimidine-synthetic pathway (Fig. 1). The reversible reaction catalyzed by DHOase is pH-dependent (Christopherson & Jones, 1979
; Porter et al.
). The biosynthetic direction (cyclization of l
-CA-asp to l
-DHO) is favoured at lower pH, while the degradative rate (l
-DHO to l
-CA-asp) is maximal at alkaline pH.
Cyclization of N-carbamyl-l-aspartate (l-CA-asp) to l-dihydroorotate (l-DHO).
DHOase belongs to the amidohydrolase superfamily, which comprises a variety of hydrolytic enzymes of the (β/α)8
-barrel (or TIM-barrel) fold (Holm & Sander, 1997
). The active sites of enzymes belonging to the amidohydrolase superfamily have five highly conserved metal-binding residues, usually four histidine residues and one aspartate residue, and one or two metal ions. A common feature of the enzyme mechanism is the utilization of an activated water or hydroxide molecule bound to the metal ion(s) at the catalytic centre.
A phylogenetic analysis of the amino-acid sequences of DHOase reveals that the enzyme can be divided into two major classes that have diverged from a common ancestor of the amidohydrolase superfamily (Fields et al.
). Type I DHOases are the more ancient form and are found in all domains of life; they include the DHOase domain of mammalian CAD and monofunctional DHOases found in Gram-positive bacteria, including Bacillus
. Mammalian CAD is a trifunctional enzyme consisting of the first three enzyme activities in the pyrimidine-synthetic pathway: carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and DHOase (Simmer et al.
; Williams et al.
). Type II DHOases are smaller (~38 kDa compared with ~45 kDa for the type I DHOases) monofunctional enzymes that are found predominantly in Gram-negative bacteria (e.g. Escherichia coli
) and have a low level of sequence identity to their type I counterparts.
To date, the structures of two DHOases, those from E. coli
and Aquiflex aeolicus
, have been reported [PDB codes 1j79
(Thoden et al.
; Lee et al.
) and 1xrt
(Martin et al.
)]. E. coli
DHOase is a monofunctional and homodimeric enzyme. Although the E. coli
DHOase has been reported to contain one catalytic zinc per monomer (Brown & Collins, 1991
; Washabaugh & Collins, 1984
), the structure clearly showed that the active site contains a binuclear centre with a carboxylated lysine as one of the bridging ligands. In the original structure determination of E. coli
DHOase, the crystals grown in the presence of racemic substrate, d
-CA-asp, were orthorhombic with a dimer in the asymmetric unit. Interestingly, one subunit contained bound l
-DHO and the other contained l
-CA-asp (Thoden et al.
In our subsequent study of E. coli
DHOase, we found two different conformations of a surface loop comprised of residues 105–115 (Lee et al.
). We also found asymmetry between the active sites in the dimer, with the product l
-DHO in one subunit and the substrate l
-CA-asp in the other, despite the fact that we used the product rather than the substrate to form the complex. However, we were able to resolve the positions of residues (109–112 from chain B
) that were missing from the original structure and to observe two conformations of the surface loop (residues 105–115). The ability to resolve the surface loops in our structure was attributed to the use of optically pure l
-DHO rather than racemic d
-CA-asp in the crystallization medium. The loop asymmetry mirrored that of the active-site contents of the two subunits. In the substrate-bound subunit the surface loop (residues 105–115) reaches in towards the active site and makes two hydrogen-bonding interactions with the bound substrate molecule via
two threonine residues (Thr109 and Thr110; ‘loop-in’), whereas the loop forms part of the surface of the protein in the product-bound subunit (‘loop-out’). Subsequent enzyme kinetics at low concentrations of l
-DHO in the reverse degradative reaction showed positive cooperativity between the subunits.
Conformational changes during enzyme catalysis have been observed in many different enzymes and appear to be a general feature of enzymatic mechanisms (Gutteridge & Thornton, 2004
; Hammes, 2002
; Kempner, 1993
). One of the movements involved in enzyme catalysis is the rearrangement of loops that constitute active-site lids. The conformations of these loops are tightly coupled to the catalytic state of the enzyme. In general, these movements are characterized by a closing of the active site, with the surface-loop regions moving in towards the active site of the protein, closing over the bound substrate. Catalysis takes place in the closed form and the enzyme opens again to release the product. This motion from open to closed is thought to fulfil a number of roles in enzyme reactions: (i) arrangement of the catalytic residues into the correct orientation for catalysis and/or restriction of the conformational freedom of the substrate, (ii) prevention of the escape of reaction intermediates before the reaction has completed and (iii) restriction of the entry of water and its subsequent reaction with unstable reaction intermediates.
To probe the role of the surface-loop movement of E. coli DHOase in catalysis, we generated a series of single-point mutants. The two threonine residues (Thr109 and Thr110) that interact with the bound substrate l-CA-asp in the active site of the wild-type enzyme were mutated to a number of different amino acids. The mutation of these residues produced enzymes with lower catalytic activities (unpublished work). In this paper, we report the crystal structure of one of the single-point mutants (T109S) of E. coli DHOase in complex with a product mimic, FOA (Fig. 2). We also discuss the behaviour of crystals of the mutant enzyme in the presence of l-DHO, which reveals a macroscopic effect of the loop movement on catalysis and crystallization.
Structure of 5-fluoroorotate (FOA).