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 His6-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a sol...
| Authors: | , , , |
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| Format: | article |
| Status: | Versión aceptada para publicación |
| Publication Date: | 2007 |
| Country: | España |
| Institution: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repository: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/45449 |
| Online Access: | http://hdl.handle.net/10261/45449 |
| Access Level: | Open access |
| Keyword: | p-coumaric acid decarboxylase Lactobacillus plantarum |
| Summary: | 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 His6-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 P43, 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 |
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