Abstract
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 28.9U/mg, which is 80-fold higher than that reported previously. Maximum activity of 18U/mg was obtained at 3-HPA concentration of 7mM, above which substrate inhibition was observed. Substrate inhibition was also seen with coenzyme A at a concentration above 0.5mM and with NADP+ above 9mM. A structure of PduP is proposed based on homology modeling. In silico docking of the co-factors coenzyme A and NAD+, respectively, showed a common binding site consisting of amino acids Thr145, Ile275, Cys277 and Ser417, which through site-directed mutagenesis to alanine and kinetic studies, were confirmed as essential for the catalytic activity of PduP.
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