Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans
| Autor: | Arun Goyal, Kedar Sharma, Aruna Rani, Arun Dhillon |
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| Rok vydání: | 2018 |
| Předmět: |
Models
Molecular 0301 basic medicine Stereochemistry Mutant Molecular Conformation Ab initio Biochemistry Substrate Specificity Catalysis Structure-Activity Relationship 03 medical and health sciences chemistry.chemical_compound Structural Biology Amino Acid Sequence Molecular Biology Protein secondary structure Binding Sites Chondroitin Lyases 030102 biochemistry & molecular biology Small-angle X-ray scattering Circular Dichroism Isothermal titration calorimetry Sequence Analysis DNA General Medicine Recombinant Proteins Enzyme Activation 030104 developmental biology chemistry Docking (molecular) Mutation Mutagenesis Site-Directed Spectrophotometry Ultraviolet Pedobacter Protein Binding Chondroitin AC lyase |
| Zdroj: | International Journal of Biological Macromolecules. 109:980-991 |
| ISSN: | 0141-8130 |
| DOI: | 10.1016/j.ijbiomac.2017.11.087 |
| Popis: | The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α)6 incomplete toroidal fold and a layered β sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% β sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×105) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during β-elimination. |
| Databáze: | OpenAIRE |
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