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Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10082-7. Epub 2005 Jul 11.

Evolution of highly active enzymes by homology-independent recombination.

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  • 1Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.


The theta-class GST enzymes hGSTT1-1 (human GSTTheta-1-1) and rGSTT2-2 (rat GSTTheta-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/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/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."

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