Crystallization and preliminary X-ray studies of Trp138Phe/Val185Thr xylose isomerases from Thermotoga neapolitana and Thermoanaerobacterium thermosulfurigenes

Xylose isomerases from Thermotoga neapolitana (TNXI) and Thermoanaerobacter ium thermosulfurigenes (TTXI) share 70.4% sequence identity and are thermos table. The double mutants Trp138Phe/Val185Thr of TNXI and TTXI have higher catalytic efficiencies than TNXI and TTXI, respectively. The Trp138Phe/ Val 185Thr TNXI and TTXI mutants were overexpressed in Escherichia coli strain BL21(DE3) and purified. Crystals of the two proteins were grown with polyet hylene glycol 8000 as the major precipitant by the hanging-drop vapour-diff usion method. Crystals of the TNXI mutant were obtained in the absence of s ubstrate, in complex with glucose and in complex with fructose. Crystals of the TTXI mutant were obtained complexed with glucose. Diffraction data wer e collected at 1.9, 2.1 and 2.1 Angstrom resolution for the fructose-TNXI m utant, glucose-TNXI mutant and substrate-unbound TNXI mutant, respectively. The diffraction data for the glucose-TTXI mutant were collected at 2.0 Ang strom resolution. The crystals belong to the orthorhombic space groups C222 (1) (TNXI mutant) and P2(1)2(1)2(1) (TTXI mutant). The TNXI and TTXI mutant crystals contain two and four monomers in the asymmetric unit, respectivel y.