Identification of glycosyltransferases mediating 2‐O‐arabinopyranosyl and 2‐O‐galactosyl substitutions of glucuronosyl side chains of xylan

• Published in: The Plant journal : for cell and molecular biology 2024-10, Vol.120 (1), p.234-252
• Main Authors: Zhong, Ruiqin, Zhou, Dayong, Phillips, Dennis R., Adams, Earle R., Chen, Lirong, Rose, John P., Wang, Bi‐Cheng, Ye, Zheng‐Hua
• Format: Article
• Language: English
• Subjects: Amino acids; Aquatic plants; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; arabinopyranosyltransferase; Arabinose - metabolism; Catalytic activity; cell wall; Cell Wall - enzymology; Cell Wall - metabolism; Cell walls; Chemical reactions; Duckweed; Enzymatic activity; Eucalyptus; Floating plants; Flowers & plants; galactosyltransferase; glucuronic acid; Glycosyltransferase; Glycosyltransferases - chemistry; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; GT47; Hemicellulose; Methyltransferase; Molecular chains; Molecular docking; Mutagenesis; Plant species; Residues; Xylan; Xylans - metabolism
• ISSN: 0960-7412; 1365-313X; 1365-313X
• DOI: 10.1111/tpj.16983

SUMMARY Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O‐2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2‐O‐arabinopyranose (Arap) or 2‐O‐galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3‐clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP‐Arap onto O‐2 of GlcA side chains of xylan, and thus it was named xylan 2‐O‐arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T‐DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon‐scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2‐O‐Arap and 2‐O‐Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site‐directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2‐O‐arabinopyranosylation and 2‐O‐galactosylation of GlcA side chains of xylan. Significance Statement Xylan is one of the major hemicelluloses in plant cell walls and its GlcA side chains may be further substituted with 2‐O‐Arap or 2‐O‐Gal residues. We have uncovered the functions of a subgroup of MUR3‐clade GT47 members in catalyzing the transfer of 2‐O‐Arap and/or 2‐O‐Gal onto GlcA side chains of xylan, which expands our understanding of glycosyltransferases involved in xylan substitutions.