PDF(Pubmed)
Abstract：
OBJECTIVE: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase.
RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.
RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.
摘要：
目的：需要新的表征的碳水化合物活性酶作为工具来区分复杂的碳水化合物结构特征。真菌糖苷水解酶家族3（GH3）β-木糖苷酶已显示可用于葡糖醛酸（GlcA）和阿拉伯呋喃糖（Araf）取代的寡木糖苷的结构阐明。来自细菌Segatellabarnioi（基本名称为Prevotellabryantii）的这些GH3真菌酶的同源物，Xyl3C,以前被描述过，但这些研究并未涉及重要的功能特异性特征.为了将该酶用于旨在区分取代的木寡糖的非还原末端的结构的实验室方法，我们进一步表征了这种GH3木糖苷酶。
结果:除了验证该木糖苷酶的基本功能特征之外,我们还确定了其作用模式,因为其涉及来自GlcA和Araf取代的寡木糖苷的非还原性末端木糖释放。Xyl3C从β-1,4-木聚糖的非还原末端切割木糖,直到出现倒数第二个取代的木糖。如果这个取代是O2连接的，然后Xyl3C除去非还原性木糖以留下取代的木糖作为新的非还原性末端。然而,如果取代是O3连接的，Xyl3C不水解，因此从非还原末端留下一个木糖(倒数第二个)的取代。因此，Xyl3C能够区分在非还原端的倒数第二个木糖上的O2和O3连接的取代。这些发现使用同样来自S.barnioi的同源酶进行对比，Xyl3B,无论存在哪个GlcA或Araf取代，都会产生倒数第二个取代的非还原末端。
结果:除了验证该木糖苷酶的基本功能特征之外,我们还确定了其作用模式,因为其涉及来自GlcA和Araf取代的寡木糖苷的非还原性末端木糖释放。Xyl3C从β-1,4-木聚糖的非还原末端切割木糖,直到出现倒数第二个取代的木糖。如果这个取代是O2连接的，然后Xyl3C除去非还原性木糖以留下取代的木糖作为新的非还原性末端。然而,如果取代是O3连接的，Xyl3C不水解，因此从非还原末端留下一个木糖(倒数第二个)的取代。因此，Xyl3C能够区分在非还原端的倒数第二个木糖上的O2和O3连接的取代。这些发现使用同样来自S.barnioi的同源酶进行对比，Xyl3B,无论存在哪个GlcA或Araf取代，都会产生倒数第二个取代的非还原末端。
