Pressure‐induced activation of latent dihydroorotase from Aquifex aeolicus as revealed by high pressure protein crystallography

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. It was previously shown that high pressure fully restores the activity of this isolated DHOase. On the basis of kinetic studies, site‐directed mutagenesis and the use of peptides mimicking loop A, a loop that appears to block access to the active site, was proposed that this pressure‐induced reactivation was due to the decrease in the volume of the system, −ΔV, resulting from the disruption of known ionic interactions between the loop and the main part of the protein. In this study, this interpretation is more precisely demonstrated by the determination of the crystallographic structure of isolated DHOase under pressure. In addition to the loop displacements, pressure induces a discrete rearrangement of the catalytic site aspartate 305, an effect that might additionally contribute to the reactivation of this enzyme. High‐pressure X‐ray crystallography in diamond anvil cell reveals how the isolated dihydroorotase from the hyperthermophilic bacterium Aquifex aeolicus is reactivated by shifting mobile loops at the surface of the catalytic centre, and by reconnecting the key residue Asp 305 to the catalytic zinc atom.