Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases

Sayaka Hayashi, Satoshi Akanuma, Wakana Onuki, Chihiro Tokunaga, Akihiko Yamagishi

Research output: Contribution to journalArticle

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Abstract

Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD +, suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.

Original languageEnglish
Pages (from-to)8583-8593
Number of pages11
JournalBiochemistry
Volume50
Issue number40
DOIs
Publication statusPublished - 2011 Oct 11
Externally publishedYes

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Coenzymes
Oxidoreductases
Substitution reactions
Temperature
Hot Temperature
Thermus thermophilus
Adenine
3-Isopropylmalate Dehydrogenase
Thermodynamic stability
Enzymes
Enzymes and Coenzymes
Mutagenesis
Denaturation
Amino Acid Substitution
Catalysis
L-Lactate Dehydrogenase
Codon
NAD
Thermodynamic properties
Modulation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases. / Hayashi, Sayaka; Akanuma, Satoshi; Onuki, Wakana; Tokunaga, Chihiro; Yamagishi, Akihiko.

In: Biochemistry, Vol. 50, No. 40, 11.10.2011, p. 8583-8593.

Research output: Contribution to journalArticle

Hayashi, Sayaka ; Akanuma, Satoshi ; Onuki, Wakana ; Tokunaga, Chihiro ; Yamagishi, Akihiko. / Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases. In: Biochemistry. 2011 ; Vol. 50, No. 40. pp. 8583-8593.
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