Aquifex aeolicus dihydroorotase: association with aspartate transcarbamoylase switches on catalytic activity

The Journal of Biological Chemistry
Anupama AhujaDavid R Evans

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 degrees 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 Clas...Continue Reading

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Citations

Jan 6, 2006·Archives of Microbiology·Donald McPhail, Margaret Shepherdson
Aug 28, 2013·Journal of Molecular Evolution·Matthieu BarbaBernard Labedan
Jun 14, 2012·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Jacqueline VitaliMichael J Colaneri
Dec 10, 2013·BMC Biochemistry·Brian F P EdwardsDavid R Evans
Dec 18, 2013·Structure·Araceli Grande-GarcíaSantiago Ramón-Maiques
Dec 20, 2013·Protein Science : a Publication of the Protein Society·Hedeel Guy EvansDavid R Evans
Apr 14, 2005·Journal of Molecular Biology·Philip D MartinBrian F P Edwards
Apr 20, 2006·Molecular and Biochemical Parasitology·Sonia M Robles LopezBarbara H Zimmermann
Dec 10, 2013·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Alba Ruiz-RamosSantiago Ramón-Maiques
Oct 21, 2016·The Journal of Biological Chemistry·Guy HervéDavid R Evans
Nov 13, 2012·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Nada LallousSantiago Ramón-Maiques

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