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.

木葡聚糖内转糖基酶/水解酶（XTH）是植物细胞壁重塑的关键酶。先前对被子植物毛白杨的研究鉴定出 11 个具有 XTH 催化基序的 PtoXTH 蛋白。令人惊讶的是，只有两个（PtoXTH27 和 PtoXTH34）表现出木葡聚糖内转葡糖基酶 (XET) 活性，表明 XTH 蛋白内存在额外的关键底物结合或催化活性位点。为了检验这一假设，我们对 PtoXTH27 和 PtoXTH34 中的选定氨基酸进行了定点诱变，并表征了它们的生化特性。分子对接模拟和生化分析表明，N101 是关键的底物结合位点，而 Q80、M81 和 V180 是影响 XTH 蛋白中 XET 活性的重要功能残基。值得注意的是，XET 缺陷的 PtoXTH26 在定点突变后恢复了活性，强调了这些残基的重要性。将我们的研究扩展到裸子植物Larix kaempferi ，我们鉴定了两种 XTH 蛋白：LkXTH1，它表现出关键底物结合位点和 XET 活性，以及​​ LkXTH2，它缺乏 XET 活性。有趣的是，LkXTH2 在定点突变后恢复了 XET 活性，突显了新鉴定的底物结合位点在不同木本植物亚门中的多功能性。该研究为快速鉴定木本植物XTH基因家族酶活性成员提供了理论基础。此外，我们的研究结果为 XTH 酶的靶向工程提供了见解，为植物生物技术和作物改良的进步铺平了道路。