Lys(13) plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearotherm ophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His(12)- Lys(13) pair instead of the Asn(12)-Gly(13) pair normally present in mesophilic TIMs. His(12) in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys(13) is unknown. To investig ate the role of the His(12) and Lys(13) pair in the enzyme's thermoadaptati on, we reintroduced the "mesophilic residues" Asn and Gly into both thermop hilic TIMs. Neither double mutant displayed diminished structural stability , but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting t hat the presence of the His(12) and Lys(13) cannot be systematically correl ated to thermoadaptation in TIMs, We determined the crystal structure of th e bTIM double mutant complexed with 2-phosphoglycolate to 2,4-Angstrom reso lution. A molecular dynamics simulation showed that upon substitution of Ly s(13) to, Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys(10). This suggests that Lys( 13) in bTIM plays a crucial role in the functional adaptation of this enzym e to high temperature. Analysis of bTIM single mutants supports this assump tion.