Xyloglucan endotransglycosylase/hydrolase (XTH) is a key enzyme in plant cell wall remodeling. Previous investigations on the angiosperm Populus tomentosa identified 11 PtoXTH proteins with the XTH catalytic motif. Surprisingly, only two (PtoXTH27 and PtoXTH34) exhibited xyloglucan endotransglucosylase (XET) activity, suggesting the presence of additional key substrate binding or catalytic activity sites within XTH proteins. To test this hypothesis, we performed site-directed mutagenesis on selected amino acids in PtoXTH27 and PtoXTH34 and characterized their biochemical properties. Molecular docking simulations and biochemical analyses revealed that N101 is a critical substrate binding site, while Q80, M81, and V180 are essential functional residues influencing XET activity in XTH proteins. Notably, XET-deficient PtoXTH26 regained activity following site-directed mutation, underscoring the significance of these residues. Expanding our study to the gymnosperm Larix kaempferi, we identified two XTH proteins: LkXTH1, which exhibited both the key substrate binding site and XET activity, and LkXTH2, which lacked XET activity. Intriguingly, LkXTH2 regained XET activity after site-directed mutation, highlighting the versatility of the newly identified substrate binding site across different woody plants subphyla. This research provides a theoretical basis for the rapid identification of enzymatically active members within the XTH gene family in woody plants. Furthermore, our findings offer insights into the targeted engineering of XTH enzymes, paving the way for advancements in plant biotechnology and crop improvement.
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