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Abstract：
Consensus design (CD) is a representative sequence-based protein design method that enables the design of highly functional proteins by analyzing vast amounts of protein sequence data. This study proposes a partial consensus design (PCD) of a protein as a derivative approach of CD. The method replaces the target protein sequence with a consensus sequence in a secondary-structure-dependent manner (i.e., regionally dependent and divided into α-helix, β-sheet, and loop regions). In this study, we generated several artificial partial consensus l-threonine 3-dehydrogenases (PcTDHs) by PCD using the TDH from Cupriavidus necator (CnTDH) as a target protein. Structural and functional analysis of PcTDHs suggested that thermostability would be independently improved when consensus mutations are introduced into the loop region of TDHs. On the other hand, enzyme kinetic parameters (kcat/Km) and average productivity would be synergistically enhanced by changing the combination of the mutations-replacement of one region of CnTDH with a consensus sequence provided only negative effects, but the negative effects were nullified when the two regions were replaced simultaneously. Taken together, we propose the hypothesis that there are protein regions that encode individual protein properties, such as thermostability and activity, and that the introduction of consensus mutations into these regions could additively or synergistically modify their functions.
摘要：
共有设计(CD)是一种代表性的基于序列的蛋白质设计方法,其能够通过分析大量的蛋白质序列数据来设计高功能的蛋白质。这项研究提出了蛋白质的部分共识设计（PCD）作为CD的衍生方法。该方法以二级结构依赖性方式用共有序列替换靶蛋白序列（即，区域依赖并分为α-螺旋，β-sheet,和循环区域)。在这项研究中,我们使用Cupriavidusnecator的TDH（CnTDH）作为靶蛋白，通过PCD产生了几种人工部分共有1-苏氨酸3-脱氢酶（PcTDHs）。PcTDHs的结构和功能分析表明，当将共有突变引入TDHs的环区时，热稳定性会得到独立改善。另一方面,酶动力学参数（kcat/Km）和平均生产率将通过改变CnTDH的一个区域的突变替换与共有序列的组合而协同增强，但是当两个区域同时替换时，负面影响就消失了。一起来看,我们提出了这样的假设，即存在编码单个蛋白质特性的蛋白质区域，如热稳定性和活性，并且将共有突变引入这些区域可以相加或协同地修饰它们的功能。
