Asparagine Tautomerization in Glycosyltransferase Catalysis. The Molecular Mechanism of Protein O-Fucosyltransferase 1.
Asparagine Tautomerization in Glycosyltransferase Catalysis. The Molecular Mechanism of Protein O-Fucosyltransferase 1.
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DOI：
10.1021/acscatal.1c01785
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发表时间：
2021-08-06
期刊：
影响因子：
12.9
通讯作者：
Rovira C
中科院分区：
文献类型：
--
作者：
Piniello B;Lira-Navarrete E;Takeuchi H;Takeuchi M;Haltiwanger RS;Hurtado-Guerrero R;Rovira C
O-糖基化是生命所必需的蛋白质翻译后修饰。参与该过程的酶之一是蛋白质O-岩藻糖基转移酶1（POFUT 1），其使表皮生长因子样结构域（EGF-LD）的特定序列背景内的苏氨酸或丝氨酸残基岩藻糖基化。与大多数转化糖基转移酶不同，POFUT 1在活性位点中缺乏碱性残基，该碱性残基可以在化学反应期间作为催化碱使EGF-LD受体的Thr/Ser残基去质子化。使用量子力学/分子力学（QM/MM）方法对最近的晶体结构，以及诱变实验，我们发现的酶催化机制，揭示它涉及质子穿梭通过活性位点天冬酰胺，物种之间保守，经历互变异构化。这一机制与秀丽隐杆线虫POFUT 1 Asn 43突变体的实验动力学分析一致，该突变体即使突变为Asp（转化糖基转移酶中的典型催化碱基）也能消除酶活性。这些结果将有助于Notch相关O-糖基化障碍的抑制剂开发。
O-glycosylation is a post-translational protein modification essential to life. One of the enzymes involved in this process is protein O-fucosyltransferase 1 (POFUT1), which fucosylates threonine or serine residues within a specific sequence context of epidermal growth factor-like domains (EGF-LD). Unlike most inverting glycosyltransferases, POFUT1 lacks a basic residue in the active site that could act as a catalytic base to deprotonate the Thr/Ser residue of the EGF-LD acceptor during the chemical reaction. Using quantum mechanics/molecular mechanics (QM/MM) methods on recent crystal structures, as well as mutagenesis experiments, we uncover the enzyme catalytic mechanism, revealing that it involves proton shuttling through an active site asparagine, conserved among species, which undergoes tautomerization. This mechanism is consistent with experimental kinetic analysis of Caenorhabditis elegans POFUT1 Asn43 mutants, which ablate enzyme activity even if mutated to Asp, the canonical catalytic base in inverting glycosyltransferases. These results will aid inhibitor development for Notch-associated O-glycosylation disorders.
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DOI：
10.1107/s2053230x18004788
发表时间：
2018-08-01
期刊：
Acta crystallographica. Section F, Structural biology communications
影响因子：
--
作者：
Lira-Navarrete E;Hurtado-Guerrero R
通讯作者：
Hurtado-Guerrero R
影响因子：
2.3
作者：
Constantino, E;Solans-Monfort, X;Bertran, J
通讯作者：
Bertran, J
影响因子：
12.9
作者：
Darby, John F.;Gilio, Amelia K.;Wu, Liang
通讯作者：
Wu, Liang
影响因子：
12.9
作者：
Nin-Hill, Alba;Rovira, Carme
通讯作者：
Rovira, Carme
DOI：
10.1073/pnas.202427399
发表时间：
2002-10-01
影响因子：
11.1
作者：
Laio, A;Parrinello, M
通讯作者：
Parrinello, M