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Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases

机译：酵母Crh1和Crh2转糖基酶的结构和功能分析

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© 2014 FEBS. Covalent cross-links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β-1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three-dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3-1,4-β-d-glucanase, followed by site-directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β-1,3-glucan, β-1,6-glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate-binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate-binding module (CBM18) of unknown function. Modeling and functional analysis of site-directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.
机译：©2014 FEBS。酵母细胞壁上几丁质和葡聚糖之间的共价交联是通过冗余蛋白Crh1和Crh2的转糖基酶活性产生的，并裂解了几丁质骨架的β-1,4键，并转移了含有新产生的还原端的分子到葡聚糖受体上。通过基于细菌1,3-1,4-β-d-d-葡聚糖酶的晶体结构的同源性建模，生成了Crh1的三维结构，然后进行定点诱变以获得有关这些酶如何实现催化作用的分子见解。通过测量表达这些蛋白质不同版本的酵母细胞对衍生自β-1,3-葡聚糖，β-1,6-葡聚糖的低聚糖进行糖基化的能力，可以表征参与其催化和结合活性的两种蛋白质的残基。和几丁质到细胞壁上的几丁质。在催化位点内，Crh1的残基E134和E138以及Crh2的E166和E170（分别对应于亲核试剂和通用酸/碱）以及Crh1和Crh2的辅助D136和D168被证明是必不可少的用于催化。位于Crh1模型的与碳水化合物结合的裂隙中的芳香族残基F152，Y160和W219的突变也影响转糖基酶活性。与Crh1不同，Crh2包含功能未知的推定碳水化合物结合模块（CBM18）。对这种CBM的定点突变残基进行建模和功能分析，确定了蛋白质折叠和稳定性必需的氨基酸，以及调节Crh2催化活性的残基。

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