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

Protein Eng Des Sel. 2016 Dec;29(12):557-562. doi: 10.1093/protein/gzw039. Epub 2016 Aug 29.


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.

Keywords: active site; chiral amine; coupled assay; fusion protein; substrate channeling.

MeSH terms

  • Actinomycetales / enzymology
  • Alcohols / metabolism*
  • Amines / metabolism*
  • Catalytic Domain
  • Models, Molecular
  • Mutation
  • Oxidoreductases / chemistry
  • Oxidoreductases / metabolism*
  • Paracoccus denitrificans / enzymology
  • Protein Engineering / methods*
  • Protein Folding
  • Protein Multimerization
  • Protein Structure, Quaternary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism*
  • Transaminases / genetics*


  • Alcohols
  • Amines
  • Recombinant Fusion Proteins
  • Oxidoreductases
  • levodione reductase
  • Transaminases