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Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations

Enzyme and Microbial Technology
Publication Date
DOI: 10.1016/j.enzmictec.2009.04.007
  • Mesophilic Adhs
  • Thermophilic Adh
  • Thermal Inactivation
  • Thermostability
  • Proline
  • Multimeric Proteins
  • Biology


Abstract The present study demonstrates the comparative thermal, conformational and kinetic stabilities of the three closely related enzymes; the mesophilic yeast alcohol dehydrogenase (YADH), horse liver alcohol dehydrogenase (HLADH), and the extreme-thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). The mid-point unfolding temperatures for TBADH and HLADH were at least 10 °C and 6 °C higher, respectively, than that of YADH. When YADH was completely inactivated by thermal stress, the residual activities of HLADH and TBADH were 70% and 100%, respectively. The optimum temperature ( T opt) activities of HLADH and TBADH were at least 40 °C and 55 °C higher, respectively, than that of YADH. Due to the higher rigidity of HLADH and TBADH, the enzymatic activation energies of HLADH and TBADH were higher than that of YADH. Geometric X-ray analysis indicated a comparatively higher coil (turn and loop) percentage in TBADH and HLADH than in YADH. Pairwise alignment for TBADH/HLADH exhibited a similarity score approximately 2.5-fold greater than that of the TBADH/YADH pair. Multiple alignments made with ClustalW revealed a higher number of conserved proline residues in the two most stable enzymes (HLADH/TBADH). These extra prolines tend to occur in surface loops and are likely to be responsible for the increased stability of TBADH and HLADH, by loop rigidification.

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