Regiospecific Acetylation of Xylan is Mediated by a Group of DUF231-Containing O-Acetyltransferases

Xylan is a major hemicellulose in the secondary walls of vessels and fibers, and its acetylation is essential for normal secondary wall assembly and properties. The acetylation of xylan can occur at multiple positions of its backbone xylosyl residues, including 2-O-monoacetylation, 3-O-monoacetylation, 2,3-di-O-acetylation and 3-O-acetylation of 2-O-glucuronic acid (GlcA)-substituted xylosyl residues, but the biochemical mechanism controlling the regiospecific acetylation of xylan is largely unknown. Here, we present biochemical characterization of a group of Arabidopsis thaliana DUF231-containing proteins, namely TBL28, ESK1/TBL29, TBL30, TBL3, TBL31, TBL32, TBL33, TBL34 and TBL35, for their roles in catalyzing the regiospecific acetylation of xylan. Acetyltransferase activity assay of recombinant proteins demonstrated that all of these proteins possessed xylan acetyltransferase activities catalyzing the transfer of acetyl groups from acetyl-CoA onto xylooligomer acceptors albeit with differential specificities. Structural analysis of their reaction products revealed that TBL28, ESK1, TBL3, TBL31 and TBL34 catalyzed xylan 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation with differential positional preference, TBL30 carried out 2-O- and 3-O-monoacetylation, TBL35 catalyzed 2,3-di-O-acetylation, and TBL32 and TBL33 mediated 3-O-acetylation of 2-O-GlcA-substituted xylosyl residues. Furthermore, mutations of the conserved GDS and DXXH motifs in ESK1 were found to result in a complete loss of its acetyltransferase activity. Together, these results establish that these nine DUF231-containing proteins are xylan acetyltransferases mediating the regiospecific acetylation of xylan and that the conserved GDS and DXXH motifs are critical for their acetyltransferase activity.