The Evolution of Catalytic Efficiency and Substrate Promiscuity in Human Theta Class 1-1 Glutathione Transferase

Abstract

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.

Ensemble Perspective for Catalytic Promiscuity

“…In these cases, the promiscuous interaction of a single enzyme or protein with multiple substrates or partners represents an evolutionary end point; the optimal function of the protein includes substrate promiscuity. Promiscuity may also be a useful trait of evolutionary intermediates that facilitates the appearance of new enzymes from an existing pool of scaffolds (3,(15)(16)(17)(18)(19)(20). In this case, mutation of substrate-specific enzymes yields promiscuous intermediates, from which new substrate specificity evolves efficiently.…”

Laboratory evolution of one disulfide isomerase to resemble another

“…Analysis of those gain-of-function mutants can provide information about functional differences between the two family members. This technique has been used to alter the enzymatic activity of a number of proteins so that they now have common properties, including the E. coli paralogs aspartate aminotransferase (AATase) and tyrosine aminotransferase (3), the structurally similar HisA and TrpF (4), and the human class 1-1 and rat class 2-2 glutathione transferases (5). Directed evolution has also been used to explore the differences between various E. coli folding catalysts, such as DsbC and DsbA (6), Grx3 and Grx1 (7), and TrxA and DsbA (8).…”

Protein Fragment Swapping: A Method for Asymmetric, Selective Site-Directed Recombination

“…Protein recombination constructs libraries of hybrids by recombining fragments from two or more parents, with the goal of discovering hybrids with beneficial properties such as improved thermostability, activity, or substrate specificity [1][2][3][4][5][6][7][8][9][10][11][12][13]. For example, Stemmer demonstrated the development of beta-lactamase hybrids with a 32,000-fold increase in the required minimum inhibitory concentration of the antibiotic cefotaxime [1].…”

“…Antibodies have long been humanized this way, e.g., combining murine variable regions with human constant regions [14,15]. An approach for the much more difficult task of humanizing enzymes (which lack the overtly modular nature of antibodies) was recently demonstrated [10,11], introducing activity from a rat glutathione transferase into a human one.…”