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

Richard D Bunker, James MJ Dickson, Tom T. Caradoc-Davies, Kerry M. Loomes, Edward N Baker

Research output: Contribution to journalArticleResearchpeer-review

4 Citations (Scopus)

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 P31 or P32 with unit-cell parameters a = b = 101.87, c = 158.85 Å.

Original languageEnglish
Pages (from-to)1259-1262
Number of pages4
JournalActa Crystallographica. Section F: Structural Biology Communications
Volume68
Issue number10
DOIs
Publication statusPublished - Oct 2012
Externally publishedYes

Keywords

  • d-xylulokinases
  • d-xylulose
  • d-xylulose 5-phosphate
  • glucuronate-xylulose pathway
  • lipogenesis
  • pentose phosphate pathway

Cite this

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title = "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 {\AA} resolution from a crystal belonging to the trigonal space group P31 or P32 with unit-cell parameters a = b = 101.87, c = 158.85 {\AA}.",
keywords = "d-xylulokinases, d-xylulose, d-xylulose 5-phosphate, glucuronate-xylulose pathway, lipogenesis, pentose phosphate pathway",
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Use of a repetitive seeding protocol to obtain diffraction-quality crystals of a putative human D-xylulokinase. / Bunker, Richard D; Dickson, James MJ; Caradoc-Davies, Tom T.; Loomes, Kerry M.; Baker, Edward N.

In: Acta Crystallographica. Section F: Structural Biology Communications, Vol. 68, No. 10, 10.2012, p. 1259-1262.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Baker, Edward N

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