کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2187958 1096147 2008 15 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Sensitive Genome-Wide Screen for Low Secondary Enzymatic Activities: The YjbQ Family Shows Thiamin Phosphate Synthase Activity
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیولوژی سلول
پیش نمایش صفحه اول مقاله
Sensitive Genome-Wide Screen for Low Secondary Enzymatic Activities: The YjbQ Family Shows Thiamin Phosphate Synthase Activity
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Molecular Biology - Volume 376, Issue 3, 22 February 2008, Pages 839–853
نویسندگان
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