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

Börner, Tim LU ; Rämisch, Sebastian LU ; Reddem, Eswar R.; Bartsch, Sebastian; Vogel, Andreas; Thunnissen, Andy Mark W.H.; Adlercreutz, Patrick LU and Grey, Carl LU (2017) In ACS Catalysis 7(2). p.1259-1269
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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
keywords
aggregation, amine transaminase, pyridoxamine 5-phosphate, stability, substrate-induced inactivation, unfolding
in
ACS Catalysis
volume
7
issue
2
pages
11 pages
publisher
The American Chemical Society
external identifiers
  • scopus:85026707057
  • wos:000393539200038
ISSN
2155-5435
DOI
10.1021/acscatal.6b02100
language
English
LU publication?
yes
id
d747967a-d7b3-4bcd-b54d-db213738edd5
date added to LUP
2017-09-01 10:50:40
date last changed
2018-04-01 04:34:58
@article{d747967a-d7b3-4bcd-b54d-db213738edd5,
  abstract     = {<p>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.</p>},
  author       = {Börner, Tim and Rämisch, Sebastian and Reddem, Eswar R. and Bartsch, Sebastian and Vogel, Andreas and Thunnissen, Andy Mark W.H. and Adlercreutz, Patrick and Grey, Carl},
  issn         = {2155-5435},
  keyword      = {aggregation,amine transaminase,pyridoxamine 5-phosphate,stability,substrate-induced inactivation,unfolding},
  language     = {eng},
  month        = {02},
  number       = {2},
  pages        = {1259--1269},
  publisher    = {The American Chemical Society},
  series       = {ACS Catalysis},
  title        = {Explaining Operational Instability of Amine Transaminases : Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability},
  url          = {http://dx.doi.org/10.1021/acscatal.6b02100},
  volume       = {7},
  year         = {2017},
}