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

Börner, Tim LU ; Rämisch, Sebastian LU ; Bartsch, Sebastian; Vogel, Andreas; Adlercreutz, Patrick LU and Grey, Carl LU (2017) In ChemBioChem
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... (More)

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.

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Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
Amines, Enzyme catalysis, Operational stability, Pyridoxamine 5′-phosphate, Transaminase
in
ChemBioChem
publisher
John Wiley & Sons
external identifiers
  • scopus:85020430872
  • wos:000406932800004
ISSN
1439-4227
DOI
10.1002/cbic.201700236
language
English
LU publication?
yes
id
d99f3583-b612-43fc-8ed6-e23670d09fdc
date added to LUP
2017-06-30 11:11:42
date last changed
2017-09-18 11:37:06
@article{d99f3583-b612-43fc-8ed6-e23670d09fdc,
  abstract     = {<p>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.</p>},
  author       = {Börner, Tim and Rämisch, Sebastian and Bartsch, Sebastian and Vogel, Andreas and Adlercreutz, Patrick and Grey, Carl},
  issn         = {1439-4227},
  keyword      = {Amines,Enzyme catalysis,Operational stability,Pyridoxamine 5′-phosphate,Transaminase},
  language     = {eng},
  month        = {06},
  publisher    = {John Wiley & Sons},
  series       = {ChemBioChem},
  title        = {Three in One : Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase},
  url          = {http://dx.doi.org/10.1002/cbic.201700236},
  year         = {2017},
}