J Biol Chem, Vol. 274, Issue 27, 19181-19187, July 2, 1999

Lys¹³ Plays a Crucial Role in the Functional Adaptation of the Thermophilic Triose-phosphate Isomerase from Bacillus stearothermophilus to High Temperatures

Marco Alvarez, Johan Wouters^¶, Dominique Maes, Véronique Mainfroid, Françoise Rentier-Delrue, Lode Wyns, Eric Depiereux^¶, and Joseph A. Martial

From the  Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, B6, Sart Tilman, B4000 Liège, Belgium, ^¶ Laboratoire de Biologie Moléculaire Structurale, Facultés Universitaires Notre-Dame de la Paix, B5000 Namur, Belgium, and  Ultrastructure Unit, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, B1640 Sint-Genesius-Rode, Belgium

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His¹²-Lys¹³ pair instead of the Asn¹²-Gly¹³ pair normally present in mesophilic TIMs. His¹² in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys¹³ is unknown. To investigate the role of the His¹² and Lys¹³ pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic 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 that the presence of the His¹² and Lys¹³ cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-Å resolution. A molecular dynamics simulation showed that upon substitution of Lys¹³ to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys¹⁰. This suggests that Lys¹³ in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.