Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.
Appl Environ Microbiol. 2012 Feb;78(3):768-77. doi: 10.1128/AEM.07031-11. Epub 2011 Dec 2.
The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to plant biomass utilization.
产热纤维梭菌属包含能够以植物多糖为食的极端嗜热细菌。该属物种的基因组揭示了某些具有独特特征的表面层同源（SLH）结构域蛋白，这些蛋白表明其在木质纤维素解构中发挥作用。本研究从这一角度对产热纤维梭菌（Caldicellulosiruptor saccharolyticus）中的两种 SLH 结构域蛋白（Csac_0678 和 Csac_2722）进行了研究。除了三个连续的 SLH 结构域外，Csac_0678 基因编码一个糖苷水解酶家族 5（GH5）催化结构域和一个家族 28 碳水化合物结合模块（CBM）；在所有测序的产热纤维梭菌属物种中都可以鉴定到与 Csac_0678 同源的基因。重组 Csac_0678 在 75°C 和 pH 值 5.0 时表现出最佳活性，同时具有内切葡聚糖酶和木聚糖酶活性。去除 SLH 结构域并不影响 Csac_0678 的 GH 活性，但缺失 CBM28 结构域则会消除对结晶纤维素的结合，并使该酶在该底物上失去活性。Csac_2722 是产热纤维梭菌基因组中最大的开放阅读框（ORF）（预测分子量为 286516 kDa），包含两个假定的糖结合结构域、两个 Big4 结构域（具有免疫球蛋白 [Ig]-样折叠的细菌结构域）和一个钙粘蛋白样（Cd）结构域。缺乏 SLH 和 Cd 结构域的重组 Csac_2722 可与纤维素结合，并具有可检测的羧甲基纤维素（CMC）水解活性。针对 Csac_0678 和 Csac_2722 的抗体证实这些蛋白结合到产热纤维梭菌的 S-层。它们的细胞定位和功能生化特性表明 Csac_0678 和 Csac_2722 参与了复杂多糖的募集和水解以及木质纤维素生物质的解构。此外，这些结果表明，其他产热纤维梭菌属基因组中的相关 SLH 结构域蛋白可能也是植物生物质利用的重要贡献者。
