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Efficient O-Glycosylation of Triterpenes Enabled by Protein Engineering of Plant Glycosyltransferase UGT74AC1
ACS Catalysis ( IF 11.3 ) Pub Date : 2020-03-04 , DOI: 10.1021/acscatal.9b05232 Jiao Li 1, 2 , Jiangang Yang 1 , Shicheng Mu 1 , Na Shang 1 , Cui Liu 1 , Yueming Zhu 1 , Yi Cai 1 , Pi Liu 1 , Jianping Lin 1 , Weidong Liu 1 , Yuanxia Sun 1, 2 , Yanhe Ma 1
ACS Catalysis ( IF 11.3 ) Pub Date : 2020-03-04 , DOI: 10.1021/acscatal.9b05232 Jiao Li 1, 2 , Jiangang Yang 1 , Shicheng Mu 1 , Na Shang 1 , Cui Liu 1 , Yueming Zhu 1 , Yi Cai 1 , Pi Liu 1 , Jianping Lin 1 , Weidong Liu 1 , Yuanxia Sun 1, 2 , Yanhe Ma 1
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Triterpene O-glycosylation has attracted significant interest from the pharmaceutical industry as a valuable means for drug design and development. Plant glycosyltransferases, which catalyze this glycosylation reaction, play a key step in preparing structure diverse and valuable triterpene glycosides. However, this class of enzymes usually suffers from low catalytic efficiency. To address this problem, triterpene glycosyltransferase UGT74AC1 from Siraitia grosvenorii was chosen and its crystal structure was solved and employed as the molecular basis to implement directed evolution and sequence/structure-based engineering. Several resultant uridine diphosphate (UDP) glycosyltransferases (UGTs) variants exhibit a 102- to 104-fold improvement in catalytic efficiency for triterpene glycosylation. Especially, one variant exhibited up to 4.17 × 104-fold increase in catalytic efficiency toward mogrol and 1.53 × 104-fold increase to UDP-glucose, respectively. Moreover, the mutants also displayed extended substrate promiscuity compared with wild-type enzyme and conserved regioselectivity. Based on the results of molecular docking and molecular dynamics simulations, it was proposed that the improved enzymatic activity and substrate promiscuity were likely owing to the stable hydrophobic interactions and favorite conformations between the enzyme and substrates. This work has also laid a foundation for the engineering of other plant UGTs for their practical application to the synthesis of valuable triterpene saponins.
中文翻译：
通过植物糖基转移酶UGT74AC1的蛋白质工程实现三萜的有效O-糖基化
三萜O-糖基化作为药物设计和开发的一种有价值的方法已经引起了制药工业的极大兴趣。催化这种糖基化反应的植物糖基转移酶在制备结构多样且有价值的三萜糖苷中起关键作用。但是，这类酶通常遭受低催化效率的困扰。为了解决这个问题，选择了罗汉果（Siraitia grosvenorii）的三萜糖基转移酶UGT74AC1，并解决了其晶体结构，并将其用作分子基础，以进行定向进化和基于序列/结构的工程。几种合成的尿苷二磷酸（UDP）糖基转移酶（UGT）变体显示10 2-至10 4三萜糖基化的催化效率提高了两倍。尤其是，一种变体对莫格罗的催化效率提高了4.17×10 4倍，而对Mogrol的催化效率提高了1.53×10 4-分别增加至UDP-葡萄糖。此外，与野生型酶相比，该突变体还显示出延长的底物混杂性和保守的区域选择性。基于分子对接和分子动力学模拟的结果，有人提出，由于稳定的疏水相互作用和酶与底物之间的偏好构象，可能提高了酶的活性和底物的混杂性。这项工作还为其他植物UGT的工程化应用奠定了基础，这些实际应用可用于合成有价值的三萜皂苷。
更新日期：2020-03-04
中文翻译：
通过植物糖基转移酶UGT74AC1的蛋白质工程实现三萜的有效O-糖基化
三萜O-糖基化作为药物设计和开发的一种有价值的方法已经引起了制药工业的极大兴趣。催化这种糖基化反应的植物糖基转移酶在制备结构多样且有价值的三萜糖苷中起关键作用。但是，这类酶通常遭受低催化效率的困扰。为了解决这个问题，选择了罗汉果（Siraitia grosvenorii）的三萜糖基转移酶UGT74AC1，并解决了其晶体结构，并将其用作分子基础，以进行定向进化和基于序列/结构的工程。几种合成的尿苷二磷酸（UDP）糖基转移酶（UGT）变体显示10 2-至10 4三萜糖基化的催化效率提高了两倍。尤其是，一种变体对莫格罗的催化效率提高了4.17×10 4倍，而对Mogrol的催化效率提高了1.53×10 4-分别增加至UDP-葡萄糖。此外，与野生型酶相比，该突变体还显示出延长的底物混杂性和保守的区域选择性。基于分子对接和分子动力学模拟的结果，有人提出，由于稳定的疏水相互作用和酶与底物之间的偏好构象，可能提高了酶的活性和底物的混杂性。这项工作还为其他植物UGT的工程化应用奠定了基础，这些实际应用可用于合成有价值的三萜皂苷。
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