The purification and preliminary X-ray diffraction studies of recombinant Synechococcus ribulose-1,5-bisphosphate carboxylase/oxygenase from Escherichia coli.
X-ray crystallographic diffraction data has been collected for recombinant hexadecameric ribulose-P2 carboxylase from the cyanobacterium Synechococcus PCC6301 expressed in Escherichia coli. The enzyme has been purified and then crystallized in a number of crystal forms from polyethylene glycol solutions. The best crystals were obtained with enzyme that was first activated with the cofactors CO2 and Mg2+ in the presence of the tight-binding intermediate analogue, 2'-carboxyarabinitol 1,5-bisphosphate. One crystal form with plate-like morphology diffracts beyond 2.5 A but has one axis greater than 350 A. A second crystal form that diffracts to similar resolution grows with space group P212121 and unit cell dimensions of a = 223.9 A, b = 111.9 A, and c = 199.7 A. The crystal forms used to collect the diffraction data have been redissolved to determine that the recombinant ribulose-P2 carboxylase L8S8 molecule is indeed composed of equal numbers of large and small subunits and also that a quaternary complex between activated ribulose-P2 carboxylase E.CO2.Mg2+, and the analogue was present in the crystals. Denaturation of the redissolved enzyme in the absence of thiol-reducing agents established that the L-subunits of the L8 core are substantially dimeric, cross-linked by a disulfide bridge. Crystals of spinach ribulose-P2 carboxylase were likewise analyzed to show that dimers of the L-subunit were also predominant. This report identifies a single cysteine residue in the L-subunit that forms a bridge between those L-monomers that compose the four putative functional dimers of the L8 core.
Newman
J
J
Department of Molecular Biology, Uppsala Biomedical Centre, Sweden.
Gutteridge
S
S
D002852:Chromatography, Ion Exchange; D003460:Crystallization; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D046911:Macromolecular Substances; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014961:X-Ray Diffraction
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase