Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus

摘要

L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gelfiltration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 angstrom resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P2(1), with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 angstrom, beta = 90.4 degrees. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a V-M value of 3.2 angstrom(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 angstrom resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 angstrom, beta = 97.38 degrees. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a V-M value of 4.0 angstrom(3) Da(-1) and a solvent content of 69%.