Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase
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 a...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society
2012
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| Online Access: | http://hdl.handle.net/1721.1/69549 https://orcid.org/0000-0002-8277-5226 |
| _version_ | 1826190767191752704 |
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| author | Puthenveetil, Sujiet Liu, Daniel S. White, Katharine Alice Thompson, Samuel M. Ting, Alice Y. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Puthenveetil, Sujiet Liu, Daniel S. White, Katharine Alice Thompson, Samuel M. Ting, Alice Y. |
| author_sort | Puthenveetil, Sujiet |
| collection | MIT |
| description | 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 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−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 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. |
| first_indexed | 2024-09-23T08:45:03Z |
| format | Article |
| id | mit-1721.1/69549 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:45:03Z |
| publishDate | 2012 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | mit-1721.1/695492022-09-30T10:59:15Z Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase Puthenveetil, Sujiet Liu, Daniel S. White, Katharine Alice Thompson, Samuel M. Ting, Alice Y. Massachusetts Institute of Technology. Department of Chemistry Ting, Alice Y. Ting, Alice Y. Puthenveetil, Sujiet Liu, Daniel S. White, Katharine Alice Thompson, Samuel M. 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 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−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 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. 2012-03-01T19:00:24Z 2012-03-01T19:00:24Z 2009-10 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/69549 Puthenveetil, Sujiet et al. “Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase.” Journal of the American Chemical Society 131.45 (2009): 16430–16438. https://orcid.org/0000-0002-8277-5226 en_US http://dx.doi.org/10.1021/ja904596f Journal of the American Chemical Society Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society Prof. Ting via Erja Kajosalo |
| spellingShingle | Puthenveetil, Sujiet Liu, Daniel S. White, Katharine Alice Thompson, Samuel M. Ting, Alice Y. Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase |
| title | Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase |
| title_full | Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase |
| title_fullStr | Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase |
| title_full_unstemmed | Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase |
| title_short | Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase |
| title_sort | yeast display evolution of a kinetically efficient 13 amino acid substrate for lipoic acid ligase |
| url | http://hdl.handle.net/1721.1/69549 https://orcid.org/0000-0002-8277-5226 |
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