Differences in protein structural regions that impact functional specificity in GT2 family β-glucan synthases

Most cell wall and secreted β-glucans are synthesised by the CAZy Glycosyltransferase 2 family (www.cazy.org), with different members catalysing the formation of (1,4)-β-, (1,3)-β-, or both (1,4)- and (1,3)-β-glucosidic linkages. Given the distinct physicochemical properties of each of the resultant β-glucans (cellulose, curdlan, and mixed linkage glucan, respectively) are crucial to their biological and biotechnological functions, there is a desire to understand the molecular evolution of synthesis and how linkage specificity is determined. With structural studies hamstrung by the instability of these proteins to solubilisation, we have utilised in silico techniques and the crystal structure for a bacterial cellulose synthase to further understand how these enzymes have evolved distinct functions. Sequence and phylogenetic analyses were performed to determine amino acid conservation, both family-wide and within each sub-family. Further structural analysis centred on comparison of a bacterial curdlan synthase homology model with the bacterial cellulose synthase crystal structure, with molecular dynamics simulations performed with their respective β-glucan products bound in the trans-membrane channel. Key residues that differentially interact with the different β-glucan chains and have sub-family-specific conservation were found to reside at the entrance of the trans-membrane channel. The linkage-specific catalytic activity of these enzymes and hence the type of β-glucan chain built is thus likely determined by the different interactions between the proteins and the first few glucose residues in the channel, which in turn dictates the position of the acceptor glucose. The sequence-function relationships for the bacterial β-glucan synthases pave the way for extending this understanding to other kingdoms, such as plants.

绝大多数细胞壁与分泌型β-葡聚糖均由CAZy糖基转移酶2家族（CAZy Glycosyltransferase 2 family，www.cazy.org）催化合成，该家族不同成员可分别催化形成(1,4)-β-、(1,3)-β-，或同时催化(1,4)-与(1,3)-β-糖苷键。鉴于最终生成的三类β-葡聚糖（分别为纤维素、热凝多糖（curdlan）与混合键型葡聚糖）各自独特的理化性质对其生物学与生物技术功能至关重要，学界亟需阐明其合成的分子演化机制，以及糖苷键连接特异性的决定方式。由于这些蛋白质的可溶性较差，阻碍了结构生物学研究的开展，本研究采用计算机模拟（in silico）技术与细菌纤维素合酶的晶体结构，进一步揭示这类酶演化出差异化催化功能的机制。本研究通过序列分析与系统发育分析，明确了该家族整体及各亚家族内的氨基酸保守性特征。后续结构分析聚焦于将细菌热凝多糖合酶的同源建模模型与细菌纤维素合酶的晶体结构进行比对，并针对结合在跨膜通道内的各自β-葡聚糖产物开展分子动力学模拟。研究发现，可与不同β-葡聚糖链产生差异化相互作用、且具有亚家族特异性保守性的关键氨基酸残基，均位于跨膜通道的入口处。因此，这类酶的连接特异性催化活性，以及最终合成的β-葡聚糖链类型，很可能由蛋白质与通道内前若干葡萄糖残基之间的相互作用差异所决定，而该相互作用又进一步决定了受体葡萄糖的位置。本研究中细菌β-葡聚糖合酶的序列-功能关联研究，为将该认知拓展至植物等其他生物界奠定了基础。