Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion

Alexandra Lerchner; Marina Daake; Alexander Jarasch; Arne Skerra

doi:10.1093/protein/gzw039

Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion

Alexandra Lerchner, Marina Daake, Alexander Jarasch, Arne Skerra

Chair of Biological Chemistry

Technical University of Munich

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

Abstract

To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT (L417M), as proven by SDS-PAGE and size exclusion chromatography. The bifunctional enzyme revealed beneficial kinetics over the two-component system, in particular at low substrate concentration.

Original language	English
Pages (from-to)	557-562
Number of pages	6
Journal	Protein Engineering, Design and Selection
Volume	29
Issue number	12
DOIs	https://doi.org/10.1093/protein/gzw039
State	Published - 2016

Keywords

Active site
Chiral amine
Coupled assay
Fusion protein
Substrate channeling

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10.1093/protein/gzw039

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abstract = "To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT (L417M), as proven by SDS-PAGE and size exclusion chromatography. The bifunctional enzyme revealed beneficial kinetics over the two-component system, in particular at low substrate concentration.",

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T1 - Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion

AU - Lerchner, Alexandra

AU - Daake, Marina

AU - Jarasch, Alexander

AU - Skerra, Arne

PY - 2016

Y1 - 2016

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AB - To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT (L417M), as proven by SDS-PAGE and size exclusion chromatography. The bifunctional enzyme revealed beneficial kinetics over the two-component system, in particular at low substrate concentration.

KW - Active site

KW - Chiral amine

KW - Coupled assay

KW - Fusion protein

KW - Substrate channeling

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Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion

Abstract

Keywords

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