Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum.
The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His(6)-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris-HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 A resolution. Crystals belonged to the tetragonal space group P4(3), with unit-cell parameters a = b = 43.15, c = 231.86 A. The estimated Matthews coefficient was 2.36 A(3) Da(-1), corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.
Rodríguez
Héctor
H
Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain.
de las Rivas
Blanca
B
Muñoz
Rosario
R
Mancheño
José M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C087194:phenylacrylic acid decarboxylase