Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state
Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state
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DOI：
10.1093/protein/gzi005
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发表时间：
2005-01-01
影响因子：
2.4
通讯作者：
Tawfik, DS
中科院分区：
文献类型：
--
作者：
Roodveldt, C;Tawfik, DS
缺陷假单胞菌磷酸三酯酶（PTE）是一种非常有效的金属酶，可水解包括有机磷神经毒剂在内的多种化合物。PTE的研究一直受到阻碍的困难，有效表达的重组形式的这种非常有趣的和潜在的有用的酶。我们确定了一个低水平的酯水解活性的PTE，然后筛选PTE基因库中的2-萘乙酸酯水解的改善。然而，试图进化这种混杂的酯酶活性导致含有三个点突变的变体（S5），其导致功能表达增加20倍。有趣的是，S5的锌全酶形式似乎比野生型PTE对热变性和添加金属螯合剂更敏感。S5变体的更高功能表达似乎在于无金属脱辅基酶的更高稳定性。在这项工作中获得的结果指出了另一个经常被忽视的蛋白质表达和纯化产量的可能决定因素，即蛋白质折叠和加工过程中中间体的稳定性。
Phosphotriesterase from Pseudomonas diminuta (PTE) is an extremely efficient metalloenzyme that hydrolyses a variety of compounds including organophosphorus nerve agents. Study of PTE has been hampered by difficulties with efficient expression of the recombinant form of this highly interesting and potentially useful enzyme. We identified a low-level esterolytic activity of PTE and then screened PTE gene libraries for improvements in 2-naphthyl acetate hydrolysis. However, the attempt to evolve this promiscuous esterase activity led to a variant (S5) containing three point mutations that resulted in a 20-fold increase in functional expression. Interestingly, the zinc holoenzyme form of S5 appears to be more sensitive than wild-type PTE to both thermal denaturation and addition of metal chelators. Higher functional expression of the S5 variant seems to lie in a higher stability of the metal-free apoenzyme. The results obtained in this work point out another-and often overlooked-possible determinant of protein expression and purification yields, i.e. the stability of intermediates during protein folding and processing.