title: Improving the NADH-cofactor specificity of the highly active AdhZ3 and AdhZ2 from Escherichia coli K-12.
abstract: Biocatalysis is a promising tool for the sustainable production of chemicals. When cofactor depending enzymatic reactions are involved the applicability of the right cofactor is a central issue. One important example in this regard is the production of alcohols by nicotinamide cofactor (NAD(P)(+)) depending alcohol dehydrogenases. AdhZ3 from Escherichia coli, which is important for the production of alcohols from biomass, has a preference for NADPH as cofactor. We used a structure guided site-specific random approach, to change the cofactor preference towards NADH and to deduce more general rules for redesigning the cofactor specificity. Transfer of a triplet motif from NADH preferring horse liver ADH to AdhZ3 showed an insufficient switch in the preference towards NADH. A combinatorial site saturation mutagenesis altering three residues at once was applied. Library screening with two different cofactor concentrations (0.1 and 0.3mM) resulted in nine improved variants with AdhZ3-LND having the highest vmax and AdhZ3-CND having the lowest K(m). Asparagine was the most frequent amino acid found in eight of nine triplet motifs. To verify the triplet-motif, two variants of E. coli AdhZ2 DIN and LND were designed and confirmed for improved activity with NADH.
authors.affiliation: Technische Universität München, Wissenschaftszentrum Straubing, Lehrstuhl für Chemie Biogener Rohstoffe, Schulgasse 16, 94315 Straubing, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Pick
authors.affiliation: Technische Universität München, Wissenschaftszentrum Straubing, Lehrstuhl für Chemie Biogener Rohstoffe, Schulgasse 16, 94315 Straubing, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Ott
authors.affiliation: Technische Universität München, Wissenschaftszentrum Straubing, Lehrstuhl für Chemie Biogener Rohstoffe, Schulgasse 16, 94315 Straubing, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Howe
authors.affiliation: Technische Universität München, Wissenschaftszentrum Straubing, Lehrstuhl für Chemie Biogener Rohstoffe, Schulgasse 16, 94315 Straubing, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Schmid
authors.affiliation: Technische Universität München, Wissenschaftszentrum Straubing, Lehrstuhl für Chemie Biogener Rohstoffe, Schulgasse 16, 94315 Straubing, Germany. Electronic address: sieber@tum.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D000426:Alcohol Dehydrogenase
keywords: AdhZ2; AdhZ3; Alcohol dehydrogenase; Cofactor; Enzyme engineering