Evolution of highly active enzymes by homology-independent recombination.
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."
Griswold
Karl E
KE
Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.
Kawarasaki
Yasuaki
Y
Ghoneim
Nada
N
Benkovic
Stephen J
SJ
Iverson
Brent L
BL
Georgiou
George
G
D000818:Animals; D003374:Coumarins; D003434:Crossing Over, Genetic / Q000235:genetics; D004976:Ethacrynic Acid / Q000378:metabolism; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
C453681:7-amino-4-chloromethylcoumarin; D003374:Coumarins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C091152:Gstt2 protein, rat; C413545:glutathione S-transferase T1; D005982:Glutathione Transferase; D004976:Ethacrynic Acid