Molecular determinants of xylose isomerase thermal stability and activity:analysis of thermozymes by site-directed mutagenesis

Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes (TTXI ) and Thermotoga neapolitana (TNXI) are 70.4% identical in their amino acid sequences and have a nearly superimposable crystal structure, Nonetheless, TNXI is much more thermostable than TTXI, Except for a few additional prol ines and fewer Asn and Gin residues in TNXI, no other obvious differences i n the enzyme structures can explain the differences in their stabilities. T NXI has two additional prolines in the Phe59 loop (Pro58 and Pro62), Mutati ons Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro in TTXI and their reverse coun terpart mutations in TNXI were constructed by site-directed mutagenesis, Su rprisingly, only the Gln58Pro mutation stabilized TTXI, The Ala62Pro and Gl n58Pro/Ala62Pro mutations both dramatically destabilized TTXI, Analysis of the three-dimensional (3D) structures of TTXI and its Ala62Pro mutant deriv ative showed a close van der Waal's contact between Pro62-C-delta and atom Lys61-C-beta (2.92 Angstrom) thus destabilizing TTXI, All the reverse count erpart mutations destabilized TNXI thus confirming that these two prolines play important roles in TNXI's thermostability, TTXI's active site has been previously engineered to improve its catalytic efficiency toward glucose a nd increase its thermostability, The same mutations were introduced into TN XI, and similar trends were observed, but to different extents, Val185Thr m utation in TNXI is the most efficient mutant derivative with a 3.1-fold inc rease in its catalytic efficiency toward glucose. With a maximal activity a t 97 degrees C of 45.4 U/mg on glucose, this TNXI mutant derivative is the most active type II XI ever reported. This 'true' glucose isomerase enginee red from a native xylose isomerase has now comparable kinetic properties on glucose and xylose.