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

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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.

Detaljer

Författare
Enheter & grupper
Externa organisationer
  • Nestlé
  • Scripps Research Institute
  • c-LEcta GmbH
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Organisk kemi

Nyckelord

Originalspråkengelska
Sidor (från-till)1482-1486
TidskriftChemBioChem
Volym18
Utgivningsnummer15
Tidigt onlinedatum2017 jun 13
StatusPublished - 2017 aug 4
PublikationskategoriForskning
Peer review utfördJa