A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro
A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro
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DOI：
10.1073/pnas.1606210113
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发表时间：
2016-09
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影响因子：
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通讯作者：
Pallinti Purushotham;Sung Hyun Cho;Sara M. Díaz-Moreno;Manish Kumar;B. Tracy Nixon;V. Bulone;J. Zimmer
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作者：
Pallinti Purushotham;Sung Hyun Cho;Sara M. Díaz-Moreno;Manish Kumar;B. Tracy Nixon;V. Bulone;J. Zimmer
意义纤维素是一种丰富的天然聚合物，主要由维管植物合成，在维管植物中形成细胞壁的承重成分。它是由膜包埋纤维素酶合成的葡萄糖分子的线性聚合物，该酶将聚合物合成与其跨质膜分泌偶联。植物表达多种纤维素合酶同种型，其被组织成不同组成的大分子组装体，这些组装体可能负责将纤维素链排列成微纤丝。在这里，我们表明，重组表达和纯化的山杨x tremuloides（杂交白杨）纤维素合成酶-8是足够的纤维素生物合成和生产纤维素微纤丝在体外。我们的研究结果表明，没有其他植物来源的因素是必需的纤维素微纤丝的生物合成。植物细胞壁是多糖、蛋白质和其他非碳水化合物聚合物的复合材料。在大多数植物组织中，最丰富的多糖是纤维素，葡萄糖分子的线性聚合物。作为细胞壁的承重组分，单个纤维素链经常被捆绑成微原纤维和大原纤维，并包裹在细胞周围。纤维素是由膜整合和进行性糖基转移酶合成的，这些糖基转移酶通过其自身的跨膜区形成的通道来消化UDP活化的葡萄糖并分泌新生聚合物。植物表达几种不同的纤维素合酶亚型在初级和次级细胞壁形成，然而，到目前为止，没有一个功能性重组在体外进行详细的生化分析。在这里，我们报告的异源表达，纯化和功能重建的山杨x tremuloides CesA 8（PttCesA 8），涉及次生细胞壁的形成。重组酶聚合UDP-活化的葡萄糖纤维素，确定通过酶降解，全甲基化糖基连接分析，电子显微镜，和诱变研究。催化活性取决于脂质双层环境和二价锰阳离子的存在。此外，电子显微镜分析显示，PttCesA 8产生几微米长的纤维素纤维，偶尔被球状颗粒覆盖，可能代表PttCesA 8复合物。酶的N-末端环指结构域的缺失几乎完全消除了纤维形成，但没有纤维素生物合成活性。我们的研究结果表明，重组PttCesA 8不仅是足够的纤维素生物合成在体外，但也足以捆绑成纤维素微纤维的个别葡聚糖链。
Significance Cellulose is an abundant natural polymer synthesized primarily by vascular plants in which it forms the load-bearing component of the cell wall. It is a linear polymer of glucose molecules synthesized by membrane-embedded cellulose synthases that couple polymer synthesis with its secretion across the plasma membrane. Plants express multiple cellulose synthase isoforms that are organized into large macromolecular assemblies of varying composition that are likely responsible for aligning the cellulose strands into microfibrils. Here we show that recombinantly expressed and purified Populus tremula x tremuloides (hybrid aspen) cellulose synthase-8 is sufficient for cellulose biosynthesis and produces cellulose microfibrils in vitro. Our results demonstrate that no other plant-derived factors are required for cellulose microfibril biosynthesis. Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.