Purification, crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate, isocitrate dehydrogenase, from Escherichia coli.
Escherichia coli isocitrate dehydrogenase (ICDH) can be phosphorylated and dephosphorylated by a single bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (AceK), which is encoded by the aceK gene. In order to investigate the regulatory mechanism of (de)phosphorylation of ICDH by AceK, AceK was successfully cocrystallized in complex with its intact protein substrate, ICDH, in the presence of ATP. The complex crystal was obtained by the hanging-drop vapour-diffusion technique using PEG 300 as a precipitant and magnesium sulfate as an additive. SDS-PAGE analysis of dissolved crystals showed that the crystals contained both AceK and ICDH proteins. The complex crystals diffracted to a resolution of 2.9 angstrom in space group P6(3), with unit-cell parameters a = b = 196.80, c = 156.46 angstrom.
Zheng
Jimin
J
Department of Biochemistry, Queen's University, Kingston, ON, Canada.
Ji
Alan Xian
AX
Jia
Zongchao
Z
D000255:Adenosine Triphosphate / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D007521:Isocitrate Dehydrogenase / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D000255:Adenosine Triphosphate; C548023:AceK protein, E coli; D007521:Isocitrate Dehydrogenase