Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase

Research output: Contribution to journalArticle

Abstract

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

Details

Authors
Organisations
External organisations
  • Nestlé Research Center
  • Scripps Research Institute
  • c-LEcta GmbH
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Organic Chemistry

Keywords

  • Amines, Enzyme catalysis, Operational stability, Pyridoxamine 5′-phosphate, Transaminase
Original languageEnglish
JournalChemBioChem
StateE-pub ahead of print - 2017 Jun 13
Peer-reviewedYes