Background

GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis.

Methods

The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74_cat) and Myceliophthora thermophila (MtXeg74A) were carried out.

Results

MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between C_α atoms of aromatic residues involved in xyloglucan binding in −3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10–12 Å. For PoGH74_cat the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft.

Conclusions

These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity.

General significance

Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.

中文翻译：

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分子动力学模拟的两个GH74内切木葡聚糖酶和突变的变体，以更好地了解酶的作用机理。

背景

GH74木葡聚糖酶由通过两个非结构化肽连接的两个单独的结构域组成。以前，有人提出结构域的移动可能影响催化酶机制的假设。

方法

的分子动力学（MD）模拟内从木葡聚糖酶-processive类芽孢杆菌odorifer（PoGH74_猫）和嗜热毁丝（MtXeg74A）中进行。

结果

与XXLG和XGXXLG寡糖复合的两种酶的MD模拟都证实了域移动的可能性。在MtXeg74A的情况下，改变C之间的距离_α参与木葡聚糖芳香残基在-3结合的原子和活性位点裂口的3子网站和那些上裂口的相对侧选择的残基的达到值高达10 –12Å。对于PoGH74_猫，构象变化不太明显。在MtXeg74A变体中，环1的缺失部分关闭了裂缝的入口，并且+3和+5亚位点的两个Trp残基额外的双重突变导致XGXXLG的迁移性增强，并且还诱导了XGXXLG的形貌变化裂。

结论

这些发现证明了GH74木葡聚糖酶以更开放和更封闭的酶构象存在的可能性。开放构象的酶可能更容易容纳分支的​​多糖，而其向封闭构象的转变以及环1的功能应有助于合成能力。

一般意义

我们的结果提供了对GH74木葡聚糖酶的作用机制的见解，并可能有助于讨论其他家族的糖苷水解酶的催化机制。