Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
J Am Chem Soc. 2009 Nov 18;131(45):16430-8. doi: 10.1021/ja904596f.
Escherichia coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine side chains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides ( Nat. Biotechnol. 2007 , 25 , 1483 - 1487 ) and photo-cross-linkers ( Angew. Chem., Int. Ed. 2008 , 47 , 7018 - 7021 ) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA's utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA's natural protein substrates have a conserved beta-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called "LplA Acceptor Peptide" (LAP). A approximately 10(7) library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a "LAP2" sequence with a k(cat)/K(m) of 0.99 muM(-1) min(-1), >70-fold better than our previous rationally designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483-1487), and only 8-fold worse than the k(cat)/K(m) values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.
大肠杆菌硫辛酸连接酶（LplA）催化三氧化代谢相关的受质蛋白上特定赖氨酸侧链与硫辛酸的共价连接。我们的实验室已经证明 LplA 和工程突变体可以取代硫辛酸，连接有用的小分子探针，如烷基叠氮化物（Nat. Biotechnol. 2007, 25, 1483-1487）和光交联剂（Angew. Chem., Int. Ed. 2008, 47, 7018-7021），从而促进成像和蛋白质组学研究。为了进一步了解硫辛酸代谢，并提高 LplA 在生物技术平台中的应用，我们设计了一种新型的 13 个氨基酸肽底物用于 LplA。LplA 的天然蛋白质底物具有保守的β发夹结构，这种构象在肽中难以重现，因此我们进行了体外进化来设计 LplA 肽底物，称为“LplA 受质肽”（LAP）。一个约 10(7)的 LAP 变体文库被展示在酵母细胞表面，用 LplA 标记硫辛酸或溴代烷酸，然后通过荧光激活细胞分选分离出标记效率最高的 LAP 克隆。经过四轮进化和额外的理性突变产生了一个“LAP2”序列，其 k(cat)/K(m)值为 0.99 muM(-1) min(-1)，比我们之前理性设计的 22 个氨基酸的 LAP1 序列（Nat. Biotechnol. 2007, 25, 1483-1487）高 70 倍，仅比天然硫辛酸和生物素受质蛋白的 k(cat)/K(m)值低 8 倍。与 LAP1 相比，动力学的改善使我们能够快速用量子点标记细胞表面肽融合受体。
