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Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases.
- Source :
-
Journal of molecular biology [J Mol Biol] 2008 Feb 01; Vol. 375 (5), pp. 1293-305. Date of Electronic Publication: 2007 Nov 12. - Publication Year :
- 2008
-
Abstract
- Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-alpha1,3-xylotriose (FAX(3)). The crystal structure of the EnXyn11A-FAX(3) complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites -3 and +2 allow accommodation of alpha1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential -3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting beta strands 11 and 12, as suggested previously, but is highly complex.
- Subjects :
- Apoenzymes chemistry
Apoenzymes isolation & purification
Avena chemistry
Binding Sites
Carrier Proteins chemistry
Carrier Proteins metabolism
Catalysis
Crystallography, X-Ray
Endo-1,4-beta Xylanases antagonists & inhibitors
Enzyme Inhibitors chemistry
Enzyme Inhibitors metabolism
Fungal Proteins antagonists & inhibitors
Fungal Proteins chemistry
Glycoside Hydrolases metabolism
Hydrogen Bonding
Hydrogen-Ion Concentration
Hydrolysis
Intracellular Signaling Peptides and Proteins
Kinetics
Models, Chemical
Models, Molecular
Mutation
Neocallimastix enzymology
Neocallimastix genetics
Neocallimastix metabolism
Penicillium enzymology
Plant Proteins chemistry
Plant Proteins metabolism
Protein Binding
Protein Conformation
Protein Folding
Protein Structure, Secondary
Structure-Activity Relationship
Substrate Specificity
Triticum enzymology
X-Ray Diffraction
Comprehension physiology
Endo-1,4-beta Xylanases chemistry
Eukaryotic Cells enzymology
Glycoside Hydrolases chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1089-8638
- Volume :
- 375
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Journal of molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 18078955
- Full Text :
- https://doi.org/10.1016/j.jmb.2007.11.007