通过改变羧基末端氨基酸残基来改造植物细胞壁修饰酶的底物特异性,The Plant Journal

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通过改变羧基末端氨基酸残基来改造植物细胞壁修饰酶的底物特异性
The Plant Journal ( IF 6.2 ) Pub Date : 2023-09-02 , DOI: 10.1111/tpj.16435
Barbora Stratilová ₁ , Sergej Šesták ₁ , Eva Stratilová ₁ , Kristína Vadinová ₁ , Stanislav Kozmon ₁ , Maria Hrmova _{2,

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Affiliation

在广泛的特异性细胞壁修饰酶（称为木葡聚糖木葡糖基转移酶（XET））中，底物识别的结构决定因素仍未得到充分定义。在这里，我们研究了Tropaeolum majus种子 TmXET6.3 同种型，它是 GH16 网络 GH16_20 亚家族的成员。该酶可识别木葡聚糖 (XG) 衍生的供体和受体，以及广泛的其他主要糖底物，尽管它缺乏同型半乳糖醛酸（果胶）片段的活性。我们专注于定义 TmXET6.3 中羧基末端残基的功能，其扩展了 GH16_20 亚家族中的受体结合区域，但在相关 GH16_21 亚家族中不存在。在羧基末端区域使用双至五重突变体进行定点诱变（在识别 XG/五半乳糖醛酸受体底物对的大麦 XET 上模拟的取代）证明可以在 TmXET6.3 中获得这种活性。我们证明了半保守的 Arg238 和 Lys237 残基的作用，在羧基末端区域引入净正电荷（与酸性五半乳糖醛酸的负电荷互补）用于木葡聚糖片段的转移。由 TmXET6.3 与 XG 寡糖供体和五半乳糖醛酸受体底物的分子模型支持的实验数据表明，它们可以容纳在活性位点中。我们的研究结果支持关于 TmXET6.3 羧基末端带正电荷残基重要性的结论，并表明可以通过受体结合位点的修饰来设计广泛的特异性。XET 中底物特异性的定义对于定义植物细胞壁的结构、动力学和功能及其代谢具有无价的价值；这些数据可适用于各种生物技术。

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Engineering of substrate specificity in a plant cell-wall modifying enzyme through alterations of carboxyl-terminal amino acid residues

Structural determinants of substrate recognition remain inadequately defined in broad specific cell-wall modifying enzymes, termed xyloglucan xyloglucosyl transferases (XETs). Here, we investigate the Tropaeolum majus seed TmXET6.3 isoform, a member of the GH16_20 subfamily of the GH16 network. This enzyme recognises xyloglucan (XG)-derived donors and acceptors, and a wide spectrum of other chiefly saccharide substrates, although it lacks the activity with homogalacturonan (pectin) fragments. We focus on defining the functionality of carboxyl-terminal residues in TmXET6.3, which extend acceptor binding regions in the GH16_20 subfamily but are absent in the related GH16_21 subfamily. Site-directed mutagenesis using double to quintuple mutants in the carboxyl-terminal region – substitutions emulated on barley XETs recognising the XG/penta-galacturonide acceptor substrate pair – demonstrated that this activity could be gained in TmXET6.3. We demonstrate the roles of semi-conserved Arg238 and Lys237 residues, introducing a net positive charge in the carboxyl-terminal region (which complements a negative charge of the acidic penta-galacturonide) for the transfer of xyloglucan fragments. Experimental data, supported by molecular modelling of TmXET6.3 with the XG oligosaccharide donor and penta-galacturonide acceptor substrates, indicated that they could be accommodated in the active site. Our findings support the conclusion on the significance of positively charged residues at the carboxyl terminus of TmXET6.3 and suggest that a broad specificity could be engineered via modifications of an acceptor binding site. The definition of substrate specificity in XETs should prove invaluable for defining the structure, dynamics, and function of plant cell walls, and their metabolism; these data could be applicable in various biotechnologies.

更新日期：2023-09-02

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