Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity.
Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity.
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对低次级酶活性进行灵敏的全基因组筛选：YjbQ 家族显示硫胺素磷酸合酶活性。
DOI：
10.1016/j.jmb.2007.12.017
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发表时间：
2008
影响因子：
5.6
通讯作者：
R. Grande
中科院分区：
文献类型：
--
作者：
E. Morett;G. Saab;L. Olvera;Maricela Olvera;H. Flores;R. Grande
当代酶是一种高效、选择性的催化剂。然而，由于活性中心本质上是非常活泼的，无端的二次反应实际上是不可避免的。因此，即使是最小的细胞，尽管其酶系有限，也有可能进行许多额外的、很可能是低效的二次反应。如果具有选择性优势，二次反应可能成为新的全功能酶进化的基础。在这里，我们调查了大肠杆菌是否具有与硫胺素生物合成相关的隐蔽酶活性。我们之所以选择这种途径，是因为这种维生素是必需的，但细胞的需求量很小。因此，活性很低的酶可以补充一些真正的硫胺素生物合成基因缺失菌株的营养缺陷症。通过过量表达大肠杆菌的蛋白质组，我们选择了yjbQ基因，该基因补充了一株缺失的硫胺素磷酸合成酶(TPS)编码基因thie。体外研究证实了TPS的活性，通过定向进化实验，这种活性得到了增强。以结构为导向的突变使我们能够确定假定的活性部位。YjbQ在Thermotoga、Sulfolobus和焦球菌中的远程同源基因被克隆，并且还显示了硫胺素营养缺乏症互补作用，这表明隐藏的TPS活性是该蛋白家族的一种特性。有趣的是，thie和yjbQ编码的TPSS是结构上没有相似性的类似酶，反映了不同的进化起源。这些结果支持这样的假设，即像大肠杆菌这样的细胞的酶制剂具有执行大量替代反应的潜力，这些反应可以被利用来进化出新的或更有效的催化剂。
Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.
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