Expression, purification, crystallization and preliminary crystallographic analysis of Trypanosoma brucei phosphofructokinase.
Phosphofructokinase from Trypanosoma brucei (TbPFK) was purified from a recombinant expression system in Escherichia coli by metal-affinity chromatography via its N-terminal His tag. The yield was 15-20 mg of pure enzyme per litre of culture. M(r) was shown to be 55 585 by mass spectrometry. Crystals suitable for X-ray diffraction analysis were obtained by the hanging-drop method of vapour diffusion with sodium formate as the precipitating agent. Monoclinic crystals of the apoenzyme grew within one week, as did orthorhombic crystals of PFK in the presence of enzymic reaction products or an active-site inhibitor. Initial attempts to solve the structure by molecular replacement with bacterial PFK structures as search models proved unrewarding, but a multiple-copy search with a polyalanine model was successful. In addition, heavy-atom soaking with platinum and mercury has yielded derivatives suitable for X-ray diffraction. A combination of the phase information from the molecular-replacement solution and the heavy-atom derivatives should allow structure solution of TbPFK. The availability of this first eukaryotic PFK structure will be of particular significance for structure-based drug design and will also provide important additional structural evidence for the allosteric control of PFK activity.
Keillor
Jeffrey W
JW
Département de Chimie, Université de Montréal, CP 6128, Succursale Centre-ville, Montréal, Québec H3C 1J7, Canada.
Lherbet
Christian
C
Castonguay
Roselyne
R
Lapierre
Danielle
D
Martinez-Oyanedel
José
J
Fothergill-Gilmore
Linda A
LA
Walkinshaw
Malcolm D
MD
D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D025221:Phosphofructokinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D025221:Phosphofructokinases