Coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from Lactobacillus reuteri: kinetic characterization and molecular modeling.
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 0.5mM and with NADP(+) above 9 mM. 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.
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Sweden. ramin.sabet_azad@biotek.lu.se
Ramin
R
Sabet-Azad
Javier A
JA
Linares-Pastén
Lisa
L
Torkelson
Roya R R
RR
Sardari
Rajni
R
Hatti-Kaul
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism; D003198:Computer Simulation; D005798:Genes, Bacterial; D007700:Kinetics; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000447:Aldehydes; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; C005556:propionaldehyde; D000445:Aldehyde Oxidoreductases; C588134:coenzyme A-acylating propionaldehyde dehydrogenase, Lactobacillus reuteri; D000444:Aldehyde Dehydrogenase; D003065:Coenzyme A
3-Hydroxypropionaldehyde; 3-Hydroxypropionic acid; Cofactor binding; Kinetic characterization; Lactobacillus reuteri; Propionaldehyde dehydrogenase