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Use of a repetitive seeding protocol to obtain diffraction-quality crystals of a putative human D-xylulokinase.


ABSTRACT: In mammals, the enzyme D-xylulokinase (XK; EC 2.7.1.17) catalyses the last step of the glucuronate-xylulose pathway, in which the ketopentose sugar D-xylulose is phosphorylated to yield D-xylulose 5-phosphate (Xu5P). Xu5P is also a metabolite of the pentose phosphate pathway and acts as a signalling molecule that regulates lipogenesis and glycolysis in the liver. To date, no eukaryotic XK has been structurally characterized. A putative human XK was expressed in Escherichia coli aided by molecular chaperones, purified and crystallized. A seeding procedure involving repeated rounds of seeding was developed and proved to be essential for obtaining diffraction-quality crystals. Preliminary X-ray diffraction analysis was performed using synchrotron radiation. This resulted in the collection of a complete diffraction data set to 2.7?Å resolution from a crystal belonging to the trigonal space group P3(1) or P3(2) with unit-cell parameters a = b = 101.87, c = 158.85?Å.

SUBMITTER: Bunker RD 

PROVIDER: S-EPMC3497992 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Use of a repetitive seeding protocol to obtain diffraction-quality crystals of a putative human D-xylulokinase.

Bunker Richard D RD   Dickson James M J JM   Caradoc-Davies Tom T TT   Loomes Kerry M KM   Baker Edward N EN  

Acta crystallographica. Section F, Structural biology and crystallization communications 20120929 Pt 10


In mammals, the enzyme D-xylulokinase (XK; EC 2.7.1.17) catalyses the last step of the glucuronate-xylulose pathway, in which the ketopentose sugar D-xylulose is phosphorylated to yield D-xylulose 5-phosphate (Xu5P). Xu5P is also a metabolite of the pentose phosphate pathway and acts as a signalling molecule that regulates lipogenesis and glycolysis in the liver. To date, no eukaryotic XK has been structurally characterized. A putative human XK was expressed in Escherichia coli aided by molecula  ...[more]

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