Evolution of highly active enzymes by homology-independent recombination

doi:10.1073/pnas.0504556102

Evolution of highly active enzymes by homology-independent recombination

Departments of ^*Chemistry and Biochemistry and ^†Chemical Engineering, and ^∥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

Abstract

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 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.”

Footnotes

↵ ** To whom correspondence may be addressed at: Department of Chemistry and Biochemistry, WEL 5.320, University of Texas, Austin, TX 78712. E-mail: biverson{at}mail.utexas.edu.
↵ †† To whom correspondence may be addressed at: Department of Chemical Engineering, CPE 4.410, University of Texas, Austin, TX 78712. E-mail: gg{at}che.utexas.edu.
↵ ‡ Present address: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
↵ § Present address: University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390.
Abbreviations: GSH, glutathione; GSTT, GSTθ; rGSTT2-2, rat GSTT2-2; hGSTT1-1, human GSTT1-1; CMAC, 7-amino-4-chloromethyl coumarin; ITCHY, incremental truncation for the creation of hybrid enzymes; C/O, crossover; r-h, rat-human; h-r, human-rat; CDNB, 1-chloro-2,4-dinitrobenzene; PEITC, phenethyl isothiocyanate.