Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase.
Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase.
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DOI：
10.1021/ja904596f
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发表时间：
2009-11-18
影响因子：
15
通讯作者：
Ting, Alice Y.
中科院分区：
文献类型：
--
作者：
Puthenveetil, Sujiet;Liu, Daniel S.;White, Katharine A.;Thompson, Samuel;Ting, Alice Y.
E.大肠杆菌硫辛酸连接酶（LplA）催化硫辛酸与参与氧化代谢的三种受体蛋白的特异性赖氨酸侧链的ATP依赖性共价连接。我们的实验室已经表明，LplA和工程化的突变体可以连接有用的小分子探针，例如烷基叠氮化物（Nat.Biotechnol. 2007，25，1483-1487）和光交联剂（Angew.化学国际艾德工程2008，47，7018-7021）代替硫辛酸，促进成像和蛋白质组学研究。为了进一步理解硫辛酸代谢，并提高LplA作为生物技术平台的效用，我们设计了一种新的LplA的13-氨基酸肽底物。LplA的天然蛋白质底物具有保守的β-发夹结构，这是一种难以在肽中重现的构象，因此我们进行了体外进化以工程化LplA肽底物，称为“LplA受体肽”（LPL）。在酵母细胞表面上展示约107个经LplA用硫辛酸或溴代链烷酸标记的BMP 1变体文库，并通过荧光激活细胞分选分离最有效标记的BMP 1克隆。四轮进化后再进行额外的合理诱变，产生了kcat/Km为0.99 μM− 1 min −1的“LAP 2”序列，比我们之前合理设计的22个氨基酸的LAP 1序列好70倍以上（Nat.Biotechnol.2004）。2007，25，1483-1487），并且仅比天然硫辛酸和生物素受体蛋白的kcat/Km值差8倍。对LAP 1的动力学改进使我们能够用量子点快速标记细胞表面肽融合受体。
E. coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine sidechains 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 photocrosslinkers (Angew. Chem Int. Ed Engl. 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 β-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 ~107 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 kcat/Km of 0.99 μM−1min−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 kcat/Km 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.
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影响因子：
6.8
作者：
Gai, S. Annie;Wittrup, K. Dane
通讯作者：
Wittrup, K. Dane
影响因子：
4.8
作者：
Fujiwara, K;Toma, S;Taniguchi, H
通讯作者：
Taniguchi, H
影响因子：
4.8
作者：
Kim, DJ;Kim, KH;Suh, SW
通讯作者：
Suh, SW
DOI：
10.1073/pnas.062171599
发表时间：
2002-05-14
影响因子：
11.1
作者：
Cummings, RT;Salowe, SP;Scolnick, EM
通讯作者：
Scolnick, EM
DOI：
10.1073/pnas.0809851105
发表时间：
2008-11-18
影响因子：
11.1
作者：
Antipov, Eugene;Cho, Art E.;Klibanov, Alexander M.
通讯作者：
Klibanov, Alexander M.