TY - JOUR
T1 - Evolution of highly active enzymes by homology-independent recombination
AU - Griswold, Karl E.
AU - Kawarasaki, Yasuaki
AU - Ghoneim, Nada
AU - Benkovic, Stephen J.
AU - Iverson, Brent L.
AU - Georgiou, George
PY - 2005/7/19
Y1 - 2005/7/19
N2 - The theta-class GST enzymes hGSTT1-1 (human GSTθ-1-1) and rGSTT2-2 (rat GSTθ-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 kcat 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."
AB - The theta-class GST enzymes hGSTT1-1 (human GSTθ-1-1) and rGSTT2-2 (rat GSTθ-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 kcat 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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U2 - 10.1073/pnas.0504556102
DO - 10.1073/pnas.0504556102
M3 - Article
C2 - 16009931
AN - SCOPUS:22544477815
SN - 0027-8424
VL - 102
SP - 10082
EP - 10087
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 29
ER -