title: Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion.
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.
authors: Munich Center for integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
authors: Alexandra
authors: A
authors: Lerchner
authors: Munich Center for integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
authors: Marina
authors: M
authors: Daake
authors: Munich Center for integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
authors: Alexander
authors: A
authors: Jarasch
authors: Munich Center for integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany skerra@tum.de.
authors: Arne
authors: A
authors: Skerra
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D000438:Alcohols / Q000378:metabolism*; D000588:Amines / Q000378:metabolism*; D020134:Catalytic Domain; D008958:Models, Molecular; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D000637:Transaminases / Q000235:genetics*
chemical_list: D000438:Alcohols; D000588:Amines; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; C437407:levodione reductase; D000637:Transaminases
keywords: active site; chiral amine; coupled assay; fusion protein; substrate channeling