Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
Daniel Ken InaokaKiyoshi Kita

Abstract

Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD-orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%.

References

Apr 28, 1968·Journal of Molecular Biology·B W Matthews
Apr 20, 2002·Current Opinion in Infectious Diseases·J A Urbina
Jul 11, 2002·Molecular and Biochemical Parasitology·Eizo TakashimaKiyoshi Kita

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Citations

Feb 28, 2008·Biochemical and Biophysical Research Communications·Matheus P PinheiroM Cristina Nonato
Mar 27, 2007·Trends in Parasitology·Kiyoshi KitaSatoshi Omura
Feb 10, 2018·Parasites & Vectors·Kartikeya Tiwari, Vikash Kumar Dubey
Nov 29, 2016·PloS One·Daniel Ken InaokaShigeharu Harada
Sep 27, 2019·European Journal of Medicinal Chemistry·Donatella BoschiMarco L Lolli

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