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内质网糖基转移酶ALG6的结构与机制
2020-02-27 16:01
来源：小柯机器人

瑞士苏黎世联邦理工学院Kaspar P. Locher课题组在研究中取得进展，他们的最新工作揭示了内质网糖基转移酶ALG6的结构与机制。该研究于2020年2月26日在线发表于《自然》杂志上。

据研究人员介绍，在真核蛋白N-糖基化过程中，一系列的糖基转移酶在将dolichylpyrophosphate连接的寡糖转移到受体蛋白之前催化其生物合成。最后的七个步骤发生在内质网腔中，需要dolichylphosphate活化的甘露糖和葡萄糖作为供体底物。负责的酶，即ALG3、ALG9、ALG12、ALG6、ALG8和ALG10，是C超家族（GT-C）的糖基转移酶，其定义为含跨膜螺旋并加工类异戊二烯连接的碳水化合物供体底物。
 
研究人员报道了了3.0Å分辨率的酵母ALG6的冷冻电镜结构，揭示了以前未知的跨膜蛋白折叠。与已报道的GT-C结构的比较表明，GT-C酶包含具有保守模块和可变模块的模块化结构，每个模块均具有不同的功能作用。研究人员使用dolichylphosphate和dolichylpyrophosphate连接糖的合成类似物以及酶促聚糖延伸来删除了供体和受体底物，这些底物利用ALG途径的纯化酶在体外重现了ALG6的活性，从而生成供体和受体底物。
 
ALG6的第二个冷冻电子显微镜结构以3.9Å的分辨率与dolichylphosphate-葡萄糖的类似物结合，揭示了该酶的活性位点。ALG6变体的功能分析确定了催化的天冬氨酸残基，其可能充当了一个通用的基础。该残基在GT-C超家族中是保守的。这些结果确定了ER-管腔GT-C酶的结构，并为理解其催化机理提供了结构基础。
 
附：英文原文
 
Title: Structure and mechanism of the ER-based glucosyltransferase ALG6

Author: Jol S. Bloch, Giorgio Pesciullesi, Jrmy Boilevin, Kamil Nosol, Rossitza N. Irobalieva, Tamis Darbre, Markus Aebi, Anthony A. Kossiakoff, Jean-Louis Reymond, Kaspar P. Locher

Issue&Volume: 2020-02-26

Abstract: In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes—ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10—are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.

DOI: 10.1038/s41586-020-2044-z

Source: https://www.nature.com/articles/s41586-020-2044-z

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
官方网址：http://www.nature.com/
投稿链接：http://www.nature.com/authors/submit_manuscript.html


本期文章：《自然》：Online/在线发表

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