Format

Send to

Choose Destination
Glycobiology. 2016 Sep;26(9):961-972. Epub 2016 May 4.

A homology model of Xyloglucan Xylosyltransferase 2 reveals critical amino acids involved in substrate binding.

Author information

1
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
2
Clemens-Schöpf-Institut for Organic Chemistry and Biochemistry, Darmstadt University of Technology, Alarich-Weiss Str. 4, 64287 Darmstadt, Germany.
3
Eduard-Zintl-Institute for Inorganic and Physical Chemistry, Darmstadt University of Technology, Alarich-Weiss-Str. 8, 64287 Darmstadt, Germany.
4
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA zabotina@iastate.edu.

Abstract

In dicotyledonous plants, xyloglucan (XyG) is the most abundant hemicellulose of the primary cell wall. The enzymes involved in XyG biosynthesis have been identified through reverse-genetics and activity was characterized by heterologous expression. Currently, there is no information on the atomic structures or amino acids involved in activity or substrate binding of any of the Golgi-localized XyG biosynthetic enzymes. A homology model of the xyloglucan xylosyltransferase 2 (XXT2) catalytic domain was built on the basis of the crystal structure of A64Rp. Molecular dynamics simulations revealed that the homology model retains the glycosyltransferase (GT)-A fold of the template structure used to build the homology model indicating that XXT2 likely has a GT-A fold. According to the XXT2 homology model, six amino acids (Phe204, Lys207, Asp228, Ser229, Asp230, His378) were selected and their contribution in catalytic activity was investigated. Site-directed mutagenesis studies show that Asp228, Asp230 and His378 are critical for XXT2 activity and are predicted to be involved in coordination of manganese ion. Lys207 was also found to be critical for protein activity and the homology model indicates a critical role in substrate binding. Additionally, Phe204 mutants have less of an impact on XXT2 activity with the largest effect when replaced with a polar residue. This is the first study that investigates the amino acids involved in substrate binding of the XyG-synthesizing xylosyltransferases and contributes to the understanding of the mechanisms of polysaccharide-synthesizing GTs and XyG biosynthesis.

KEYWORDS:

glycosyltransferase, homology modeling, plant cell wall, xyloglucan

PMID:
27146522
DOI:
10.1093/glycob/cww050
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Silverchair Information Systems
Loading ...
Support Center