Aquifex aeolicus Dihydroorotase

Abstract

Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The enzyme from Aquifex aeolicus, a hyperthermophilic organism of ancient lineage, was cloned and expressed in Escherichia coli. The purified protein was found to be a 45-kDa monomer containing a single zinc ion. Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested. However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism. Whereas the k cat of 13.8 ؎ 0.03 s ؊1 was close to the value observed for the mammalian enzyme, the K m for dihydroorotate, 3.03 ؎ 0.05 mM was 433-fold higher. Gel filtration and chemical cross-linking showed that the complex exists as a 240-kDa hexamer (DHO 3 -ATC 3 ) and a 480-kDa duodecamer (DHO 6 -ATC 6 ) probably in rapid equilibrium. Complex formation protects both DHOase and ATCase against thermal degradation at temperatures near 100°C where the organism grows optimally. These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme. CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co 2؉ -substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site. The association of DHOase with ATCase probably serves as a molecular switch that ensures that free, uncomplexed DHOase in the cell remains inactive. At pH 7.4, the equilibrium ratio of carbamoyl aspartate to dihydroorotate is 17 and complex formation may drive the reaction in the biosynthetic direction.

Dihydroorotase from the Hyperthermophile Aquifiex aeolicus Is Activated by Stoichiometric Association with Aspartate Transcarbamoylase and Forms a One-Pot Reactor for Pyrimidine Biosynthesis

“…Separately, in solution, AaeDHO is a monomer and AaeATC is a very stable trimer that can only be dissociated with denaturants (6, 37). When mixed at room temperature and neutral pH, they form a major complex of 480 kDa and a minor complex of 240 kDa.…”

“…The genes for A. aeolicus ATC and DHO were inserted into the Bam HI/ Nco I sites of plasmid pRSETC, which adds a 6×His tag fused to the N-terminal end, and expressed separately in E. coli BL21(DE3) cells (5, 6). The cells were cotransformed with the helper plasmid pSJS1240, which overexpresses tRNA species for three codons that are common in A. aeolicus but rare in E. coli.…”

“…Among these examples, A. aeolicus is notable because (a) it is one of the oldest species of eubacteria and has a closer phylogenetic connection to eukaryotes than other eubacteria, (b) it is a hyperthermophile that flourishes at 95 °C, and so its proteins are particularly stable, and (c) its separately expressed enzymes form a weak complex between CPS and ATC (5) and a strong complex between DHO and ATC (6). In this paper, we present the crystal structure of the DHO-ATC complex (DAC) from A. aeolicus at 2.3 Å resolution with adventitious citrate bound in the active site.…”

“…The trimer alone is active, but two trimers readily associate with six AaeDHO chains to form a heteromeric dodecamer with enhanced thermal stability for both components (5, 6). This partnership identifies AaeATC as a type A1 aspartate transcarbamoylase (7, 8), in which the DHO subunits are active (e.g., A. aeolicus (6), Thermus aquaticus (9)). In type A2 complexes, the DHO domain is inactive and only fulfills a structural role (e.g., Pseudomonas aeruginosa (10)).…”

“…AaeDHO is notable for being inactive unless bound to its cognate ATC, the preceding enzyme in the pathway (6). Structures of the inactive enzyme have identified three possible factors responsible for the latent state: (a) access to the active site is blocked by a bond between Znα and Cys181, which is not present in EcoDHO, (b) the enzyme lacks the second zinc atom present in EcoDHO, and (c) three loops around the periphery of the active site are intrinsically disordered (22).…”