Xyloglucan Xylosyltransferase 1 Displays Promiscuity Toward Donor Substrates During in Vitro Reactions
Xyloglucan Xylosyltransferase 1 Displays Promiscuity Toward Donor Substrates During in Vitro Reactions
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DOI：
10.1093/pcp/pcab114
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发表时间：
2021-07-15
影响因子：
4.9
通讯作者：
Zabotina, Olga A.
中科院分区：
文献类型：
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作者：
Ehrlich, Jacqueline J.;Weerts, Richard M.;Zabotina, Olga A.
糖基转移酶（GT）是一个大家族的酶，其通过利用各种各样的活化糖形式的供体底物将糖添加到广泛的受体底物，包括多糖、蛋白质和脂质。一般认为单个GT表现出高水平的底物特异性，但这尚未在非常大的GT集合中进行彻底研究。在这里，我们研究了木葡聚糖木糖基转移酶1（XXT 1），这是一种参与植物细胞壁多糖木葡聚糖合成的GT。木葡聚糖具有葡聚糖主链，具有仅由来自尿苷二磷酸（UDP）-木糖的木糖组成的初始侧链取代。虽然这种保守的取代模式表明XXT1的高底物特异性，但我们的体外动力学研究阐明了一组更复杂的行为。动力学研究表明，与UDP-木糖和UDP-葡萄糖反应的k（cat）值相当，而与UDP-阿拉伯糖和UDP-半乳糖的反应慢10倍以上。使用k（cat）/K-M作为效率的量度，UDP-木糖作为底物的效率是次佳替代物UDP-葡萄糖的8倍。据我们所知，我们是第一个证明，并非所有的植物XXTs是高度底物特异性的，有些在体外反应中确实显示出显着的混杂性。动力学参数本身可能无法解释植物中的高底物选择性，这表明在多糖生物合成过程中存在额外的控制机制。对GT底物特异性的进一步理解将有助于蛋白质工程、诊断工具的开发和对生物系统的理解。
Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate xyloglucan xylosyltransferase 1 (XXT1), a GT involved in the synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from uridine diphosphate (UDP)-xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable k(cat) values for reactions with UDP-xylose and UDP-glucose, while reactions with UDP-arabinose and UDP-galactose are over 10-fold slower. Using k(cat)/K-M as a measure of efficiency, UDP-xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting that there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems.