Crystal structure and structure-guided tunnel engineering in a bacterial β-1,4-galactosyltransferase

文献类型： 外文期刊

第一作者： Luo, Guocong

作者： Luo, Guocong;Huang, Zhaolin;Zhu, Yingying;Chen, Jiajun;Ni, Dawei;Xu, Wei;Zhang, Wenli;Mu, Wanmeng;Rao, Yijian;Hou, Xiaodong

作者机构：

关键词： Biosynthetic LNnT; Tunnel engineering; beta-1,4-galactosyltransferase; Biosynthetic LNnT; Tunnel engineering

期刊名称：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES （ 影响因子：8.5； 五年影响因子：8.7 ）

ISSN： 0141-8130

年卷期： 2024 年 279 卷

页码：

收录情况： SCI

摘要： Lacto-N-neotetraose (LNnT), a representative oligosaccharide found in human milk, has been previously examined for its beneficial traits. However, the LNnT titer is limited by the efficient glycosyltransferase pathway, particularly with respect to the catalysis of rate-limiting steps. As data on the crystal structure of the key enzyme required for synthesizing LNnT are lacking, the synthesis of LNnT remains an uncertainty. Here, for the first time we report the three-dimensional structure of a bacterial beta-1,4-galactosyltransferase, Aa beta 4GalT, and analyze the critical role played by residues in its catalytic efficacy. Guided by structural insights, we engineered this enzyme to enhance its catalytic efficiency using structure-guided tunnel engineering. The mutant enzyme L5 (K155M/H156D/F157W/K185M/Q216V) so produced, showed a 50-fold enhancement in catalytic activity. Crystal structure analysis revealed that the mechanism underlying the improvement in activity was of the swing door type. The closed conformation formed by dense hydrophobic packing with Q216V-K155M widened and permitted substrate entry. Our results show that altering the tunnel conformation helped appropriately accommodate the substrate for catalysis and provide a structural basis for the modification of other glycosyltransferases.

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