Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii
Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii
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DOI：
10.1128/jb.01897-14
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发表时间：
2014-08
影响因子：
3.2
通讯作者：
Hiroshi Yokoyama;T. Yamashita;R. Morioka;H. Ohmori
中科院分区：
文献类型：
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作者：
Hiroshi Yokoyama;T. Yamashita;R. Morioka;H. Ohmori
摘要在厌氧条件下，热纤维素酶能在70°C以上的高温下有效降解纤维素、木聚糖和天然草。C. besenchymal细胞外分泌多结构域糖苷水解酶沿着未知功能的蛋白质。在这项研究中，我们分析了C。通过质谱分析，我们确定了四种具有高pI值（9.2至9.6）的非催化性植物细胞壁结合蛋白（PWBP）。保守结构域数据库的搜索表明，这些蛋白质具有一个共同的结构域相关的溶质结合蛋白。另外，在C.最好的基因组序列为了分析PWBP的结合特性，制备了在大肠杆菌中表达的重组PWBP 57和PWBP 65。PWBPs显示出广泛的结合特异性：它们与纤维素、地衣聚糖、木聚糖、阿拉伯木聚糖、葡糖醛酸木聚糖、甘露聚糖、葡甘露聚糖、果胶、寡糖和梯牧草的细胞壁结合。这些蛋白质对植物细胞壁表现出最高的结合亲和力，在所测试的不溶性多糖中，结合常数（Ka）值为5.2 × 106至44 × 106 M−1，使用耗尽结合等温线测量。亲和凝胶电泳表明，蛋白质结合的酸性聚合物果胶最强烈的可溶性多糖测试。荧光显微镜分析表明，在一片草叶中，蛋白质优先结合在细胞壁上。结合具有高pI值的非催化PWBP可能是C.在高温下会产生不良反应。
ABSTRACT Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (Ka) values of 5.2 × 106 to 44 × 106 M−1 among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.