Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America ; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America ; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America ; Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
PLoS One. 2013 Dec 16;8(12):e84172. doi: 10.1371/journal.pone.0084172. eCollection 2013.
During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases produced by this hyperthermophilic bacterium.
在结晶纤维素上生长时，嗜热细菌 Caldicellulosiruptor bescii 会分泌几种纤维素降解酶。其中一种酶是 CelA（CbCel9A/Cel48A），据报道，它是该细菌中分泌量最高的纤维素酶。CbCel9A/Cel48A 是一种大型多模块多肽，由一个 N 端催化糖苷水解酶家族 9（GH9）模块和一个 C 端 GH48 催化模块组成，它们被一个家族 3c 碳水化合物结合模块（CBM3c）和两个相同的 CBM3bs 隔开。野生型 CbCel9A/Cel48A 和其截短突变体分别在巨大芽孢杆菌和大肠杆菌中表达。野生型多肽从 Avicel 中释放的葡萄糖当量是缺乏 GH48 催化模块的截短突变体的两倍。携带 GH9 模块和 CBM3c 的截短突变体比野生型蛋白更耐热，这可能是由于其紧凑的结构。主要的水解活性存在于 GH9 催化模块中，而含有 GH48 模块和三个 CBMs 的截短突变体在降解结晶或无定形纤维素时无效。有趣的是，含有 CBM3bs 的 GH9 和/或 GH48 催化模块在水解结晶纤维素时形成低密度颗粒。此外，含有 GH9 和 GH48 模块的 TM3（GH9/CBM3c）和含有三个 CBM3 模块的 TM2（GH48）协同水解结晶纤维素。CBM3bs 的缺失或使其结合活性受损的突变表明，这些 CBM 在结晶纤维素的水解过程中很重要。与这一观察结果一致，预测编码纤维素降解酶的 Caldicellulosiruptor bescii 基因簇中的九个基因中有七个含有 CBM3bs。基于我们的结果，我们假设 Caldicellulosiruptor bescii 使用 GH48 模块和 CBM3bs 在 CbCel9A/Cel48A 中使结晶纤维素的某些区域失稳，以便被该嗜热细菌产生的高度活跃的 GH9 模块和其他内切葡聚糖酶攻击。
