Elucidation of the role of Ser329 and the C-terminal region in the catalytic activity of Pseudomonas stutzeril-rhamnose isomerase
| Autor: | Kosei Takeda, Hiromi Yoshida, Shigehiro Kamitori, Ken Izumori |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Stereochemistry
Molecular Sequence Data Bioengineering Isomerase medicine.disease_cause Biochemistry Substrate Specificity Structure-Activity Relationship Residue (chemistry) Protein structure Bacterial Proteins X-Ray Diffraction Sequence Analysis Protein Catalytic Domain Escherichia coli Serine medicine Amino Acid Sequence Molecular Biology Aldose-Ketose Isomerases Hexoses L-rhamnose isomerase Pseudomonas stutzeri chemistry.chemical_classification Sequence Homology Amino Acid biology Active site Molecular Sequence Annotation biology.organism_classification Protein Structure Tertiary Amino acid chemistry Mutation Biocatalysis Mutagenesis Site-Directed biology.protein Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection. 23:919-927 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzq077 |
| Popis: | Pseudomonas stutzeri l-rhamnose isomerase (l-RhI) is capable of catalyzing the isomerization between various aldoses and ketoses, showing high catalytic activity with broad substrate-specificity compared with Escherichia coli l-RhI. In a previous study, the crystal structure of P. stutzeri l-RhI revealed an active site comparable with that of E. coli l-RhI and d-xylose isomerases (d-XIs) with structurally conserved amino acids, but also with a different residue seemingly responsible for the specificity of P. stutzeri l-RhI, though the residue itself does not interact with the bound substrate. This residue, Ser329, corresponds to Phe336 in E. coli l-RhI and Lys294 in Actinoplanes missouriensis d-XI. To elucidate the role of Ser329 in P. stutzeri l-RhI, we constructed mutants, S329F (E. coli l-RhI type), S329K (A. missouriensis d-XI type), S329L and S329A. Analyses of the catalytic activity and crystal structure of the mutants revealed a hydroxyl group of Ser329 to be crucial for catalytic activity via interaction with a water molecule. In addition, in complexes with substrate, the mutants S329F and S329L exhibited significant electron density in the C-terminal region not observed in the wild-type P. stutzeri l-RhI. The C-terminal region of P. stutzeri l-RhI has flexibility and shows a flip-flop movement at the inter-molecular surface of the dimeric form. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|