Dihydroorotase from Escherichia coli: Loop Movement and Cooperativity between Subunits

M, Lee; Chan, Camilla; Guss, J. Mitchell; Christopherson, Richard I.; Maher, M.J.

doi:10.1016/j.jmb.2005.01.067

Cited by 31 publications

(70 citation statements)

References 33 publications

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“…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, 2005). 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.…”

Section: Introductionmentioning

confidence: 71%

“…The purified protein was dialyzed into 20 mM Na HEPES pH 7.2 and 1 mM DTT. Crystals were grown by the hanging-drop vapourdiffusion method at 277 K. In the initial attempt to grow crystals of T109S DHOase, the crystallization conditions for native DHOase were applied, using 2 ml protein solution (8.2 mg ml À1 ) mixed with 2 ml reservoir solution (15-20% PEG 3350, 0.1 M MES pH 6-6.5, 75 mM MgCl 2 and 0.15 M KCl) and 0.45 ml 100 mM l-DHO (Lee et al, 2005). Crystallization conditions were modified slightly in order to increase the stability of tetragonal crystals by using reservoir solution containing 14-16% PEG 3350, 0.1 M MES pH 6.25, 25 mM 0.2 M KCl and 30% sucrose.…”

Section: Crystallizationmentioning

confidence: 99%

“…Chain A of the native DHOase structure in space group P2 1 2 1 2 1 (PDB code 1xge; Lee et al, 2005) was used as the search model. The Zn atoms, ligand molecule and solvent molecules were deleted from the search model.…”

Section: Structure Solution and Refinementmentioning

confidence: 99%

“…To date, the structures of two DHOases, those from E. coli and Aquiflex aeolicus, have been reported [PDB codes 1j79 and 1xge (Thoden et al, 2001;Lee et al, 2005) and 1xrt and 1xrf (Martin et al, 2005)]. E. coli DHOase is a monofunctional and homodimeric enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…These crystals are extremely unstable and disintegrate shortly after formation, which is followed by the growth of orthorhombic crystals from the remnants of the tetragonal crystals or at new nucleation sites. Orthorhombic crystals, for which a structure has previously been reported [Thoden et al (2001), Biochemistry, 40, 6989-6997; Lee et al (2005), J. Mol. Biol.…”

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confidence: 96%

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Structure of the T109S mutant ofEscherichia colidihydroorotase complexed with the inhibitor 5-fluoroorotate: catalytic activity is reflected by the crystal form

Maher

Guss

2007

Acta Cryst Sect F

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PDB Reference: dihydroorotase, 2e25, r2e25sf.Crystals of a single-point mutant (T109S) of Escherichia coli dihydroorotase (DHOase) with diminished activity grown in the presence of l-dihydroorotate (l-DHO) are tetragonal, with a monomer in the asymmetric unit. These crystals are extremely unstable and disintegrate shortly after formation, which is followed by the growth of orthorhombic crystals from the remnants of the tetragonal crystals or at new nucleation sites. Orthorhombic crystals, for which a structure has previously been reported [Thoden et al. (2001), Biochemistry, 40, 6989-6997; Lee et al. (2005), J. Mol. Biol. 348, 523-533], contain a dimer of DHOase in the asymmetric unit; the active site of one monomer contains the substrate N-carbamyl-l-aspartate (l-CA-asp) and the active site of the other monomer contains the product of the reaction, l-DHO. In the subunit with l-DHO in the active site, a surface loop (residues 105-115) is 'open'. In the other subunit, with l-CA-asp in the active site, the loop folds inwards, forming specific hydrogen bonds from the loop to the l-CA-asp. The tetragonal crystal form can be stabilized by crystallization in the presence of the inhibitor 5-fluoroorotate (FOA), a product (l-DHO) mimic. Crystals of the complex of T109S DHOase with FOA are tetragonal, space group P4 1 2 1 2, with unit-cell parameters a = b = 72.6, c = 176.1 Å . The structure has been refined to R and R free values of 0.218 and 0.257, despite severe anisotropy of the diffraction. In this structure, the flexible loops are both in the 'open' conformation, which is consistent with FOA, like l-DHO, binding at both sites. The behaviour of the T109S mutant crystals of DHOase in the presence of l-DHO is explained by initial binding of l-DHO to both subunits, followed by slow conversion to l-CA-asp, with consequent movement of the flexible loop and dissolution of the crystals. Orthorhombic crystals are then able to grow in the presence of l-DHO and l-CA-asp.

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Section: Introductionmentioning

confidence: 71%

Section: Crystallizationmentioning

confidence: 99%

Section: Structure Solution and Refinementmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 96%

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Structure of the T109S mutant ofEscherichia colidihydroorotase complexed with the inhibitor 5-fluoroorotate: catalytic activity is reflected by the crystal form

Maher

Guss

2007

Acta Cryst Sect F

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CAD: A Multifunctional Protein LeadingDe NovoPyrimidine Biosynthesis

Moreno-Morcillo

Ramón‐Maiques

2017

Encyclopedia of Life Sciences

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Pyrimidines are essential precursors for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) synthesis, protein glycosylation and lipid synthesis. In resting cells, pyrimidines are largely obtained through salvage pathways, but in proliferating cells, particularly in tumours, the synthesis of pyrimidines de novo is indispensable to fuel the high demand of nucleic acids and other cellular components. In animals, the de novo pathway is initiated and controlled by CAD, a ∼240‐kDa multifunctional protein with four different enzymatic domains: glutaminase (GLN), carbamoyl phosphate synthetase (CPS), dihydroorotase (DHO) and aspartate transcarbamoylase (ATC). In contrast, in bacteria, archaeans and plants, GLN, CPS, DHO and ATC are distinct monofunctional proteins. The structures of a number of these enzymes from bacteria and archaea are known, but until recently, there was no structural information about CAD other than that it self‐assembles into ∼1.5‐megaDa hexamers. Key Concepts De novo synthesis of pyrimidine nucleotides is essential for cell growth and proliferation. In animals, the multifunctional protein CAD catalyses the first three reactions of de novo pyrimidine synthesis. CAD is a 243‐kDa polypeptide with four enzymatic domains [glutaminase (GLN), carbamoyl phosphate synthetase (CPS), dihydroorotase (DHO) and aspartate transcarbamoylase (ATC)] that oligomerises into 1.5‐megaDa hexamers. In bacteria, GLN, CPS, DHO and ATC are individual proteins for which structural information is available. The crystal structures of the DHO and ATC domains of human CAD were recently reported. The GLN and CPS domains of CAD are expected to be similar to the Escherichia coli CPS and human mitochondrial CPS1 crystal structures. A model of CAD is proposed that sets the DHO and ATC domains as the central framework of the hexameric particles.

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