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Crystallization of Chlorella deoxyuridine triphosphatase.

ABSTRACT: Deoxyuridine triphosphatase (dUTPase) is a ubiquitous enzyme that has been widely studied owing to its function and evolutionary significance. The gene coding for the dUTPase from the Chlorella alga was codon-optimized and synthesized. The synthetic gene was expressed in Escherichia coli and recombinant core Chlorella dUTPase (chdUTPase) was purified. Crystallization of chdUTPase was performed by the repetitive hanging-drop vapor-diffusion method at 298 K with ammonium sulfate as the precipitant. In the presence of 2'-deoxyuridine-5'-[(?,?)-imido]triphosphate and magnesium, the enzyme produced die-shaped hexagonal R3 crystals with unit-cell parameters a = b = 66.9, c = 93.6 Å, ? = 120°. X-ray diffraction data for chdUTPase were collected to 1.6 Å resolution. The crystallization of chdUTPase with manganese resulted in very fragile clusters of needles.

SUBMITTER: Badalucco L

PROVIDER: S-EPMC3232149 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Crystallization of Chlorella deoxyuridine triphosphatase.

Badalucco Laura L Poudel Ishwari I Yamanishi Mamoru M Natarajan Chandrasekhar C Moriyama Hideaki H

Acta crystallographica. Section F, Structural biology and crystallization communications 20111126 Pt 12

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous enzyme that has been widely studied owing to its function and evolutionary significance. The gene coding for the dUTPase from the Chlorella alga was codon-optimized and synthesized. The synthetic gene was expressed in Escherichia coli and recombinant core Chlorella dUTPase (chdUTPase) was purified. Crystallization of chdUTPase was performed by the repetitive hanging-drop vapor-diffusion method at 298 K with ammonium sulfate as the precipitant ...[more]

PMID: 22139176

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Crystallization and crystal-packing studies of Chlorella virus deoxyuridine triphosphatase.

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Chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima.

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| S-EPMC29678 | biostudies-literature

Guanosine triphosphatase stimulation of oncogenic Ras mutants.

Project description:Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.

| S-EPMC22057 | biostudies-literature

The crystal structure of the Leishmania major deoxyuridine triphosphate nucleotidohydrolase in complex with nucleotide analogues, dUMP, and deoxyuridine.

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| S-EPMC3091252 | biostudies-literature

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