Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

Michika Sasaki, Mayumi Uno, Satoshi Akanuma, Akihiko Yamagishi

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the K m values for NAD (nicotinamide adenine dinucleotide) and/or the kcat values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.

Original languageEnglish
Pages (from-to)721-727
Number of pages7
JournalProtein Engineering, Design and Selection
Volume21
Issue number12
DOIs
Publication statusPublished - 2008 Dec 1

Keywords

  • Catalytic efficiency
  • Low-temperature activity
  • Random mutagenesis
  • Thermal stability
  • Thermostable enzyme

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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