Improving the NADH-cofactor specificity of the highly active AdhZ3 and AdhZ2 from Escherichia coli K-12

Bio‐based α,ω‐Functionalized Hydrocarbons from Multi‐step Reaction Sequences with Bio‐ and Metallo‐catalysts Based on the Fatty Acid Decarboxylase OleTJE

“…When comparing the influence of 10 % vs. 30 % v/v DMSO used as co‐solvent, it was found that 30 % DMSO had a negative effect on the activity of Ec AdhZ3‐LND, whereas in LCAO_ Af reactions, no difference was observed. According to reported studies, the efficiency of the LCAO_ Af reaction continuously increases from pH 6 to a maximum pH 10, whereas Ec AdhZ3‐LND has an optimal pH between 5.5 and 6 for using NAD + as cofactor . A pH of 7 appeared to offer the most favorable conditions for both LCAO_ Af and OleT.…”

“…According to reported studies, the efficiency of the LCAO_Af reaction continuously increases from pH 6t oa maximum pH 10, [24] whereas EcAdhZ3-LND hasa no ptimal pH between 5.5 and 6f or using NAD + as cofactor. [23] Ap Ho f7 appeared to offer the most favorable conditions for both LCAO_Af and OleT.…”

“…Once the decarboxylation reactiono ft he w-hydroxy fatty acid 1a was completed, the oxidation of the terminal alcohol 1b to ac arboxylic group to produce 1e via the aldehyde 1d was attempted.T wo enzymes were tested for this task:a ne nzyme variant of the NAD + -dependent alcohol dehydrogenase from E. coli (EcAdhZ3-LND)w here the SSN (serine, serine, asparagine) motif was substituted by aL ND (leucine, asparagine, aspartic acid) motif in the nucleotide-binding region, [23] and aputative flavin-dependent long-chain alcohol oxidase from Aspergillus fumigatus (LCAO_Af). [24] To determine the influenceo ft he conditions established for the decarboxylation reaction on the other enzymes of the cas-cade (Scheme 2), we investigated different buffers and co-solvents.…”

“…As eries of in vitro reactionc ascades combines the de-functionalization by enzymatic decarboxylation to ar e-functionalization of the w-hydroxy group or the double bonds (Scheme 2). The first and central step of all valorization chains is the decarboxylation of 1a by OleT to form 1b.T he further oxidation of 1b to undec-10-en-1-al (1d)c an be carried out by am utated NAD + dependent alcohol dehydrogenase from Escherichia coli (EcAdhZ3-LND) [23] or aF AD + dependent alcohol oxidase from Aspergillus fumigatus (LCAO_Af). [24] Af urthero xidation of 1d by either of the used enzymes can lead to the formation of undec-10-en-1-oic acid (1e).…”

Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity

“…Although this switch has been attempted in a number of different enzymes (see, e.g. Scrutton et al , 1990 ; Chen et al , 1995 ; Rane and Calvo, 1997 ; Lerchner et al , 2013 ; Pick et al , 2014 ), it is often accompanied by a loss in catalytic efficiency. However, a study in which engineering a ketol-acid reductoisomerase (KARI) to be NADH-dependent caused a 3-fold increase in titre yield, despite decreased activity in vitro , indicates that in vitro activity is not always an accurate representation of in vivo activity ( Bastian et al , 2011 ).…”