Explaining Operational Instability of Amine Transaminases: Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability

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Abstract

The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it to the two commonly used dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis. In the presence of substrate, all three ATAs featured reduced stability in comparison to their resting stability, but the tetramer showed slower inactivation rates than the dimeric ATAs. Kinetic and thermodynamic analysis revealed an amine donor induced inactivation mechanism involving accumulation of the less stable aminated enzyme-cofactor intermediate. Dissociation of the enzyme-PMP complex forms the unstable apoenzyme, which can rapidly unfold. Crystal structure analysis shed light on the structure-function relationship suggesting that the cofactor-ring binding element is stabilized in the quaternary structure conferring higher operational stability by minimizing PMP leakage and apoenzyme formation. In contrast to the common practice, increasing the amine acceptor content improved the stability and substrate turnover of dimeric ATAs. An extra supply of the pyridoxal cofactor (PLP) enhanced the stability of dimeric and tetrameric ATAs but reduced the transamination activity. The ATA inactivation mechanism described here provides valuable aspects for both process development and protein engineering.

Detaljer

Författare
Enheter & grupper
Externa organisationer
  • Scripps Research Institute
  • University of Groningen
  • c-LEcta GmbH
Forskningsområden

Nyckelord

Originalspråkengelska
Sidor (från-till)1259-1269
Antal sidor11
TidskriftACS Catalysis
Volym7
Utgivningsnummer2
StatusPublished - 2017 feb 3
PublikationskategoriForskning
Peer review utfördJa