Characterization of the low-temperature activity of Sulfolobus tokodaii glucose-1-dehydrogenase mutants

Taisuke Sugii, Satoshi Akanuma, Sota Yagi, Kazuki Yagyu, Yukiko Shimoda, Akihiko Yamagishi

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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°C 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.

Original languageEnglish
Pages (from-to)367-371
Number of pages5
JournalJournal of Bioscience and Bioengineering
Volume118
Issue number4
DOIs
Publication statusPublished - 2014 Oct 1
Externally publishedYes

Keywords

  • Circular dichroism
  • Enzyme activity
  • Glucose-1-dehydrogenase
  • Low-temperature activity
  • Site-directed mutagenesis
  • Steady-state kinetics
  • Thermal stability
  • Thermophilic enzyme

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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