Kapoor Divya, Kumar Vijay, Chandrayan Sanjeev K, Ahmed Shubbir, Sharma Swati, Datt Manish, Singh Balvinder, Karthikeyan Subramanian, Guptasarma Purnananda
Division of Protein Science and Engineering, Institute of Microbial Technology, Chandigarh 160 036, India.
Biochim Biophys Acta. 2008 Nov;1784(11):1771-6. doi: 10.1016/j.bbapap.2008.05.007. Epub 2008 Jun 13.
Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.
通过对主要为非连续残基进行几十次合理选择的替换、插入和缺失操作，我们对作为嗜热纤维素酶（海栖热袍菌Cel12A）底物结合和催化活性凹槽的β折叠的溶剂暴露面进行了重塑，使其在结构和功能上类似于嗜温同源物（里氏木霉Cel12A）的等效凹槽。工程化蛋白，一种中温活性-耐热纤维素酶（MT Cel12A），展现出其嗜温祖先的最佳功能温度和嗜热祖先的解链温度，这表明将嗜温来源的表面“嫁接”到嗜热来源的结构支架上，有可能帮助生成重组来自生命不同域的同源蛋白质的结构和功能特征的新型酶。
