<b>Method</b><br>Kinetic assays were performed using a photometer (Hitachi U-2000 Spectrophotometer, in SUPRASIL quartz cuvettes (path length 10 mm) at 35°C (or temperature as indicated). As assay buffer a solution of 50 mM HK2PO4/H2KPO4, 50 mM Tris/HCl, 1 mM DTT, 20% glycerol, pH 7.8 (or at pH as indicated) was used. For quantifying the oxidation (Ox) of aldehydes, each 400 µL reaction contained buffer, 2 mM NAD+ (or as indicated), 5 mM CoA (or as indicated), purified enzyme (heterologously expressed in <i>E. coli</i>, N-terminal 6xHis tag; wild type = WT, enzyme variants with A254I, A254P or A254G replacements) at concentrations as indicated and various concentrations of aldehydes. For quantifying the reduction (Red) of acyl-CoAs, each 400 µL reaction contained buffer, 0.625 mM NADH (or as indicated), purified enzyme (WT) at concentrations as indicated and various concentrations of acyl-CoAs. The enzyme activity was monitored by measuring the formation/consumption of NADH at 340 nm using an absorption coefficient of 6.22 mM-1 cm-1. Enzyme activity (U) is expressed as µmol NADH produced/consumed per minute. Steady state kinetic data, obtained with technical repeats, were analyzed by non-linear regression to the Michaelis-Menten (MM) equation using GraphPad Prism software.<br><br><b>Data</b><br>The following data sets are included in this collection:<br><br>Temperature and pH optima<br>Co-factor Km values<br>Kinetic Data for aldehydes and acyl-CoAs<br><br><b>Raw data processing method</b><br><br>The following method was used to process the data:<br><br>-Export raw data from photometer (see file Assay_Rawdata.txt and corresponding meta data file Assay_Metadata.txt)<br>-Analyze slope (Abs340nm/s) by linear regression<br>-Calculate enzyme activity in U/mg enzyme<br>-Calculate mean and SD for technical replicates<br>-Analyze by nonlinear regression (curve fit, MM). <br
