3-Hydroxypropionic acid (3-HP), an important C3 chemical for a bio-based industry, is natively produced by Lactobacillus reuteri from glycerol. Conversion of glycerol occurs via the intermediate 3-hydroxypropionaldehyde (3-HPA), followed by an ATP-producing pathway initiated by the CoA-acylating propionaldehyde dehydrogenase (PduP). The pduP gene of L reuteri was cloned and expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity for characterization of its activity and properties. Kinetic studies with propionaldehyde as substrate showed a maximum specific activity of 28.9 U/mg, which is 80-fold higher than that reported previously. Maximum activity of 18 U/mg was obtained at 3-HPA concentration of 7 mM, above which substrate inhibition was observed. Substrate inhibition was also seen with coenzyme A at a concentration above 0.5 mM and with NADP(+) above 9 m M. A structure of PduP is proposed based on homology modeling. In silica docking of the co-factors coenzyme A and NAD(+), respectively, showed a common binding site consisting of amino acids Thr145, IIe275, Cys277 and Ser417, which through site-directed mutagenesis to alanine and kinetic studies, were confirmed as essential for the catalytic activity of PduP. (c) 2013 Elsevier Inc. All rights reserved.