Crystallization and preliminary X-ray analysis of native and selenomethionine fructose-1,6-bisphosphate aldolase from Thermus aquaticus.
Fructose-1,6-bisphosphate aldolase (E.C. 4.1.2) catalyses the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in the glycolytic pathway of prokaryote and eukaryote organisms. The enzyme was obtained from the extreme thermophile Thermus aquaticus and, in contrast to mesophilic aldolases, expresses maximal activity in the presence of Co(2+) as cofactor instead of Zn(2+). The purified recombinant protein was monodisperse according to dynamic light-scattering measurements. Crystals of recombinant native class II fructose-1,6-bisphosphate aldolase from T. aquaticus were obtained from two different starting conditions at low protein concentrations. Condition I, using the sitting-drop vapour-diffusion method, yielded monoclinic crystals having space group P2 and unit-cell parameters a = 99.5, b = 57.5, c = 138.6 A, beta = 90.25 degrees. Diffraction data were collected to 2 A resolution at beamline X8-C of the NSLS synchrotron-radiation source. Native and selenomethionine-substituted protein crystals were obtained from condition II by hanging-drop vapor diffusion. The tetragonal crystals of the native protein belong to the space group P4(1), with unit-cell parameters a = b = 88.8, c = 163.1 A, while those of the SeMet protein have space group I4(1), with unit-cell parameters a = b = 88.6, c = 164.1 A. A data set suitable for MAD phasing was collected to 2.6 A resolution at beamline X8-C of the NSLS synchrotron source.
Sauvé
V
V
Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal H3C 3J7, Canada.
Sygusch
J
J
D003460:Crystallization; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology*; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D012645:Selenomethionine; D005634:Fructose-Bisphosphate Aldolase