title: Activation of Latent Dihydroorotase from Aquifex aeolicus by Pressure.
abstract: Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues.
authors.affiliation: From the Laboratoire BIOSIPE, Sorbonne Universités, Institut de Biologie Paris Seine, CNRS, Université Pierre et Marie Curie, 75005 Paris, France, guy.herve@upmc.fr.
authors.forename: Guy
authors.initials: G
authors.lastname: Hervé
authors.affiliation: the Department of Chemistry, Eastern Michigan University, Ypsilanti, Michigan 48197, and.|the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: Hedeel Guy
authors.initials: HG
authors.lastname: Evans
authors.affiliation: the Department of Chemistry, Eastern Michigan University, Ypsilanti, Michigan 48197, and.
authors.forename: Roshini
authors.initials: R
authors.lastname: Fernado
authors.affiliation: the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: Chandni
authors.initials: C
authors.lastname: Patel
authors.affiliation: the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: Fatme
authors.initials: F
authors.lastname: Hachem
authors.affiliation: the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201 ab2285@wayne.edu.
authors.forename: David R
authors.initials: DR
authors.lastname: Evans
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000502:physiology; D004080:Dihydroorotase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006874:Hydrostatic Pressure; D055503:Protein Multimerization / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase
keywords: aspartate transcarbamoylase; dihydroorotase; enzyme inactivation; extreme thermophile; hydrostatic pressure; inhibition mechanism; kinetics; protein conformation; pyrimidine biosynthesis