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

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Three in One : Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase. / Börner, Tim; Rämisch, Sebastian; Bartsch, Sebastian; Vogel, Andreas; Adlercreutz, Patrick; Grey, Carl.

In: ChemBioChem, Vol. 18, No. 15, 04.08.2017, p. 1482-1486.

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Börner, Tim ; Rämisch, Sebastian ; Bartsch, Sebastian ; Vogel, Andreas ; Adlercreutz, Patrick ; Grey, Carl. / Three in One : Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase. In: ChemBioChem. 2017 ; Vol. 18, No. 15. pp. 1482-1486.

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TY - JOUR

T1 - Three in One

T2 - ChemBioChem

AU - Börner, Tim

AU - Rämisch, Sebastian

AU - Bartsch, Sebastian

AU - Vogel, Andreas

AU - Adlercreutz, Patrick

AU - Grey, Carl

PY - 2017/8/4

Y1 - 2017/8/4

N2 - 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.

AB - 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.

KW - Amines

KW - Enzyme catalysis

KW - Operational stability

KW - Pyridoxamine 5′-phosphate

KW - Transaminase

UR - http://www.scopus.com/inward/record.url?scp=85020430872&partnerID=8YFLogxK

U2 - 10.1002/cbic.201700236

DO - 10.1002/cbic.201700236

M3 - Article

VL - 18

SP - 1482

EP - 1486

JO - ChemBioChem

JF - ChemBioChem

SN - 1439-4227

IS - 15

ER -