Journal article
Characterisation of a novel endo-xyloglucanase (XcXGHA) from Xanthomonas that accommodates a xylosyl-substituted glucose at subsite −1
Department of Chemical and Biochemical Engineering, Technical University of Denmark1
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark2
Wageningen University & Research3
University of Copenhagen4
Norwegian University of Life Sciences5
A xyloglucan-specific endo-1,4β-glucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae has been cloned, expressed in Escherichia coli, purified and characterised. The XcXGHA enzyme belongs to CAZy family GH74 and has catalytic site residues conserved with other xyloglucanases in this family.
At its optimal reaction conditions, pH 7.0 and 40 °C, the enzyme has a kcat/KM value of 2.2 × 107 min−1 M−1 on a tamarind seed xyloglucan substrate. XcXGHA is relatively stable within a broad pH range (pH 4–9) and up to 50 °C (t1/2, 50 °C of 74 min). XcXGHA is proven to be xyloglucan-specific, and a glycan microarray study verifies that XcXGHA catalyses cleavage of xyloglucan extracted from both monocot and dicot plant species.
The enzyme catalyses hydrolysis of tamarind xyloglucan in a unique way by cleaving XXXG into XX and XG (X is xylosyl-substituted glucose; G is unsubstituted glucose), is able to degrade more complex xyloglucans and notably is able to cleave near more substituted xyloglucan motifs such as L [i.e. α-l-Fucp-(1 → 2)-β-d-Galp-(1 → 2)-α-d-Xylp-(1 → 6)-β-d-Glcp].
LC-MS/MS analysis of product profiles of tamarind xyloglucan which had been catalytically degraded by XcXGHA revealed that XcXGHA has specificity for X in subsite −1. The 3D model suggests that XcXGHA consists of two seven-bladed β-propeller domains with the catalytic center formed by the interface of these two domains, which is conserved in xyloglucanases in the GH74 family.
However, the XcXGHA has two amino acids (D264 and R472) that differ from the conserved residues of other GH74 xyloglucanases. These two amino acids were predicted to be located on the opposite side of the active site pocket, facing each other and forming a closing surface above the active site pocket.
These two amino acids may contribute to the unique substrate specificity of the XcXGHA enzyme.
Language: | English |
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Publisher: | Springer Berlin Heidelberg |
Year: | 2014 |
Pages: | 9667-9679 |
ISSN: | 14320614 and 01757598 |
Types: | Journal article |
DOI: | 10.1007/s00253-014-5825-1 |
ORCIDs: | Meyer, Anne S. and Mikkelsen, Jørn Dalgaard |
Active site pocket Amino acids Biomaterials Catalysis Cleavage pattern Cleavage patterns Cloning Enzyme activity Enzymes Escherichia coli GH74 Glucose HASH(0x56acdc8) Optimal reaction condition Plant shutdowns Polymers Substituted Glcm Substrate specificity Tamarind seed xyloglucan XX bond Xyloglucanase
Biomedical and Life Sciences Biotechnology Catalytic Domain Cloning, Molecular DNA, Bacterial Enzyme Stability Gene Expression Glucans Glycoside Hydrolases Hydrogen-Ion Concentration Life Sciences Microbial Genetics and Genomics Microbiology Models, Molecular Molecular Sequence Data Protein Conformation Sequence Analysis, DNA Substrate Specificity Temperature Xanthomonas Xylans xyloglucan xyloglucan endo(1-4)-beta-D-glucanase