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Characterization of the low-temperature activity ofSulfolobus tokodaiiglucose-1-dehydrogenase mutants

Journal of Bioscience and Bioengineering
DOI: 10.1016/j.jbiosc.2014.03.002
  • Enzyme Activity
  • Circular Dichroism
  • Glucose-1-Dehydrogenase
  • Low-Temperature Activity
  • Site-Directed Mutagenesis
  • Steady-State Kinetics
  • Thermal Stability
  • Thermophilic Enzyme
  • Biology


Thermophilic enzymes are potentially useful for industrial processes because they are generally more stable than are mesophilic or psychrophilic enzymes. However, a crucial drawback for their use in such processes is that most thermophilic enzymes are nearly inactive at moderate and low temperatures. We have previously proposed that modulation of the coenzyme-binding pocket of thermophilic dehydrogenases can produce mutated proteins with enhanced low-temperature activities. In the current study, we produced and characterized mutants of an NADP-dependent glucose-1-dehydrogenase from the hyperthermophile Sulfolobus tokodaii in which a predicted coenzyme-binding, non-polar residue was replaced by another non-polar residue. Detailed analyses of the kinetic properties of the wild-type enzyme and its mutants showed that one of the mutants (V254I) had improved kcat and kcat/Km values at both 25 and 80°C. Temperature-induced unfolding experiments showed that the thermal stability of the mutant enzyme was comparable to that of the wild-type enzyme. Calculation of the energetic contribution of the V254I mutation for the dehydrogenase reaction revealed that the mutation destabilizes the enzyme-NADP+-glucose ternary complex and reduces the transition-state energy, thus enhancing catalysis.

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