research3 - Evolution of highly active enzymes by...

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Unformatted text preview: Evolution of highly active enzymes by homology-independent recombination Karl E. Griswold*, Yasuaki Kawarasaki †‡ , Nada Ghoneim †§ , Stephen J. Benkovic ¶ , Brent L. Iverson* i **, and George Georgiou † i †† Departments of *Chemistry and Biochemistry and † Chemical Engineering, and i Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712; and ¶ Department of Chemistry, Pennsylvania State University, University Park, PA 16802 Contributed by Stephen J. Benkovic, June 10, 2005 The theta-class GST enzymes hGSTT1-1 (human GST u-1-1) and rGSTT2-2 (rat GST u-2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C y Os) at positions not accessible by DNA family shuffling. High- throughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C y Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k cat with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and low- homology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme ‘‘humanization.’’ enzyme engineering u enzyme humanization u GST u ITCHY u high-throughput screening G STs play a crucial role in cellular detoxification by conju- gating reactive electrophilic compounds to the tripeptide glutathione (GSH) (1). Mammals encode at least seven distinct classes of GSTs. The theta enzymes represent the oldest class of GSTs and, as such, are produced by a surprisingly diverse array of animals, plants, algae, and bacteria (2). Although theta-class enzymes retain the highly conserved GST 3D fold, they possess a characteristic C-terminal a-helical extension, which, unlike that of some alpha-class enzymes, covers both the electrophile and GSH-binding sites, thus clearly differentiating the theta class from other members of the soluble GST superfamily (3). The rat GST u-2-2 (rGSTT2-2, 244 aa) and human GST u-1-1 (hGSTT1-1, 240 aa) enzymes exhibit 54.3% overall amino acid identity. The GSH-binding domain, or G site, of the two enzymes (residues ' 1–77) is conserved (79.2% amino acid identity), and the 5 9 regions that encode this domain (nucleotides 1–231) exhibit 74.5% DNA sequence identity. In contrast, the electro-exhibit 74....
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This note was uploaded on 12/04/2011 for the course CHEM 590A taught by Professor Staff during the Summer '10 term at University of Illinois, Urbana Champaign.

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research3 - Evolution of highly active enzymes by...

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