Sanz-Aparicio J, Hermoso J A, Martínez-Ripoll M, González B, López-Camacho C, Polaina J
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Madrid, Spain.
Proteins. 1998 Dec 1;33(4):567-76. doi: 10.1002/(sici)1097-0134(19981201)33:4<567::aid-prot9>3.0.co;2-u.
The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. beta-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (alpha/beta)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix alpha2, to Asp28, located in one of the loops surrounding the active-site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions.
生物技术产业的不断发展要求设计出适用于通常需要对不利条件具有广泛抗性的环境的酶。多粘芽孢杆菌的β-葡萄糖苷酶A是蛋白质工程研究的一个有趣模型。这是一种特征明确的酶，属于糖基水解酶家族1。其天然底物是纤维二糖，但对各种人工底物也有活性。其天然状态具有八聚体结构。其亚基保留了该家族一般的（α/β）8桶状拓扑结构，活性位点位于沿桶轴定义的腔内。通过随机诱变，我们鉴定出了几种增强其稳定性的突变，并且发现其中一个突变，即E96K替换，涉及结构变化。该突变体的晶体结构已通过X射线衍射确定，并与天然结构进行了比较。在两种结构之间发现的唯一差异是一个新的离子对，它将在α2螺旋N端引入的赖氨酸96与位于活性位点腔周围环之一中的天冬氨酸28连接起来。这个新的离子对结合了序列上相距较远的链段，因此，这种有利的相互作用必定在稳定三级结构方面发挥决定性影响。此外，对晶体学各向同性温度因子的分析表明，作为引入离子对的直接结果，观察到靠近突变位点的桶状二级结构单元出现了意外的迁移率降低。然而，这种效应仅在形成盐桥的其中一个伙伴周围观察到，而在另一个伙伴周围未观察到。这些结果表明，在蛋白质结构内单个氨基酸替换可产生深远影响。因此，鉴定和组合一些影响稳定性的单个替换可能足以获得一种高度抗性的酶，适用于极端条件下使用。
