2025/09/26 by Ruiqin Zhong, Dayong Zhou, Dennis Phillips +3 · 1 voice
Agricultural and Biological Sciences · Biochemistry, Genetics and Molecular Biology · Engineering · #Biofuel production and bioconversion #Enzyme Production and Characterization #Polysaccharides and Plant Cell Walls
paper · doi:10.1093/pcp/pcaf122
openalex publication_date 2025/09/26 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/22
Plant cell wall polysaccharide glycosyltransferases catalyze the transfer of sugars from specific nucleotide sugar donors onto specific acceptor substrates. The mechanisms of how their enzymatic specificity is determined are one of the long-standing questions in plant cell wall biology. In this report, we studied the biochemical functions of Arabidopsis and poplar GT61 glycosyltransferases involved in xylan substitutions and investigated the molecular determinants of their nucleotide sugar donor specificity. Enzymatic activity assays of recombinant proteins of Arabidopsis and poplar GT61 members demonstrated that two of them, AtX2AT1 and PtrX2AT1, exhibited xylan 2-O-arabinosyltransferase activities specifically using UDP-Araf, two other ones, AtXYXT2/3, possessed xylan 2-O-xylosyltransferase activities specifically using UDP-Xyl, and three other ones, PtrXXAT1/2/3, were able to catalyze the transfer of 2-O-Araf and 2-O-Xyl onto xylan using both UDP-Araf and UDP-Xyl. Structural modeling and molecular docking of PtrXXAT1 identified amino acid residues involved in interacting with UDP-Araf and UDP-Xyl at the putative active site and site-directed mutagenesis revealed their critical roles in PtrXXAT1 catalytic activities. Furthermore, structural alignment and reciprocal swapping of UDP-Xyl-interacting residues of PtrXXAT1 with their corresponding residues of AtX2AT1 pinpointed key residues determining their nucleotide sugar donor specificity. Our results indicate that Arabidopsis and poplar GT61 members catalyze 2-O-Araf- and/or 2-O-Xyl substitutions of xylan and that subtle structural differences in their substrate-binding pockets could alter their substrate specificity toward nucleotide sugar donors.