Engineering the substrate specificity of xylose isomerase
| Autor: | Matti Leisola, Ossi Pastinen, Tarja Parkkinen, Ossi Turunen, Juha Rouvinen, Harri Santa, Johanna Karimäki |
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| Rok vydání: | 2005 |
| Předmět: |
Xylose isomerase
Models Molecular Time Factors Stereochemistry Protein Conformation Static Electricity Bioengineering Crystallography X-Ray Protein Engineering Biochemistry Catalysis Substrate Specificity Catalytic Domain Enzyme kinetics Binding site Molecular Biology Aldose-Ketose Isomerases Binding Sites biology Hydrogen bond Chemistry Lysine Temperature Substrate (chemistry) Active site Hydrogen Bonding Ligand (biochemistry) Arabinose Streptomyces Oxygen Kinetics Glucose Models Chemical Mutation biology.protein Mutagenesis Site-Directed Thermodynamics Biotechnology |
| Zdroj: | Protein engineering, designselection : PEDS. 17(12) |
| ISSN: | 1741-0126 |
| Popis: | Xylose isomerase (XI) catalyzes the isomerization and epimerization of hexoses, pentoses and tetroses. In order to clarify the reasons for the low reaction efficiency of a pentose sugar, L-arabinose, we determined the crystal structure of Streptomyces rubiginosus XI complexed with L-arabinose. The crystal structure revealed that, when compared with D-xylose and D-glucose, L-arabinose binds to the active site in a partially different position, in which the ligand has difficulties in binding the catalytic metal M2. Lys183 has been thought to stabilize the open substrate conformation by hydrogen bonding to oxygen O1. Our results with L-arabinose showed that the substrate stays in a linear form even without a hydrogen bond between Lys183 and oxygen O1. We engineered mutations to the active site of Actinoplanes missouriensis XI to improve the reaction efficiency with L-arabinose. The mutation F26W was intended to shift the position of oxygen O1 of L-arabinose closer to the catalytic metal M2. This effect of F26W was modeled by free energy perturbation simulations. In line with this, F26W increased 2-fold the catalytic efficiency of XI with L-arabinose; the increase was seen mainly in kcat. The mutation Q256D was outside the sphere of the catalytic residues and probably modified the electrostatic properties of the active site. It improved 3-fold the catalytic efficiency of XI with L-arabinose; this increase was seen in both Km and kcat. This study showed that it is possible to engineer the substrate specificity of XI. |
| Databáze: | OpenAIRE |
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