Biochemical insights into the regiospecificity of xyloglucan galactosyltransferases

The plant cell wall hemicellulose xyloglucan in most dicot species consists of repeating units of three consecutive xylosylated Glc residues followed by an unsubstituted Glc (XXXG). Available evidence suggests that galactosylation of the second and the third Xyl side chains of XXXG is carried out regiospecifically by two xyloglucan galactosyltransferases XLT2 and MUR3, respectively, resulting in XLXG and XXLG units, but the mechanism underlying their regiospecificity remains elusive. In this report, we demonstrated that recombinant MUR3 and XLT2 proteins of Arabidopsis, poplar and duckweed were able to regiospecifically galactosylate not only XXXG, but also XLXG and XXLG, respectively, to generate XLLG. Interestingly, they were also able to galactosylate mono- and di-xylosylated xyloglucan oligomers. Protein structural modeling revealed that Arabidopsis and poplar MUR3 proteins contained an α-helical lid-like domain covering their active site clefts and its deletion led to increased galactosyltransferase activity. Molecular docking of the structural models of MUR3 and XLT2 identified amino acid residues interacting with UDP-Gal and XXXG in their active site clefts. Furthermore, site-directed mutagenesis uncovered critical roles of these substrate-interacting residues in the catalytic activity. Together, these findings provide biochemical insights into the molecular determinants of the regiospecificity of MUR3 and XLT2 in xyloglucan galactosylation.