Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity
Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity
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DOI：
10.1073/pnas.0500063102
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发表时间：
2005-05-10
影响因子：
11.1
通讯作者：
Iverson, BL
中科院分区：
文献类型：
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作者：
Varadarajan, N;Gam, J;Iverson, BL
酶的精细选择性和催化活性是在进化过程中正负选择压力的作用下形成的。相比之下，与天然对应物相比，通过使用体外筛选技术改造以接受新型底物的酶变体通常显示出更高程度的催化混杂性和更低的总周转率。使用细菌展示和多参数流式细胞术，我们已经开发了一种新的方法，用于模拟正和负选择压力在体外分离酶的变体与所需的新底物的反应性，同时排除那些对不需要的底物的反应性。筛选大肠杆菌内肽酶OmpT的随机突变体的大文库导致分离酶变体1.3.19，其切割Ala-Arg肽键而不是WT酶优选的Arg-Arg键。变体1.3.19表现出大于三百万倍的选择性（-Ala-Arg-/-Arg-Arg-）和Ala-Arg裂解的催化效率，其与亲本对优选底物Arg-Arg所表现出的催化效率相同。一个单一的氨基酸Ser 223 Arg取代显示出完全概括了1.3.19变体的独特催化性质。这些结果可以通过提出该突变起到将通常在WT底物肽中发现的P-1 Arg侧链与OmpT的S-1亚位点中的223 Arg侧链“交换”的作用来解释。
The exquisite selectivity and catalytic activity of enzymes have been shaped by the effects of positive and negative selection pressure during the course of evolution. In contrast, enzyme variants engineered by using in vitro screening techniques to accept novel substrates typically display a higher degree of catalytic promiscuity and lower total turnover in comparison with their natural counterparts. Using bacterial display and multiparameter flow cytometry, we have developed a novel methodology for emulating positive and negative selective pressure in vitro for the isolation of enzyme variants with reactivity for desired novel substrates, while simultaneously excluding those with reactivity toward undesired substrates. Screening of a large library of random mutants of the Escherichia colt endopeptidase OmpT led to the isolation of an enzyme variant, 1.3.19, that cleaved an Ala-Arg peptide bond instead of the Arg-Arg bond preferred by the WT enzyme. Variant 1.3.19 exhibited greater than three million-fold selectivity (-Ala-Arg-/-Arg-Arg-) and a catalytic efficiency for Ala-Arg cleavage that is the same as that displayed by the parent for the preferred substrate, Arg-Arg. A single amino acid Ser223Arg substitution was shown to recapitulate completely the unique catalytic properties of the 1.3.19 variant. These results can be explained by proposing that this mutation acts to "swap" the P-1 Arg side chain normally found in WT substrate peptides with the 223Arg side chain in the S-1 subsite of OmpT.