Structure-Based Redesign of GST A2-2 for Enhanced Catalytic Efficiency with Azathioprine

Abstract

Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.

Multidimensional epistasis and fitness landscapes in enzyme evolution

“…These interactions were not present in the parental enzyme A2*E. The L107G mutation appeared to contribute to a slightly narrower and more stable Hsite pocket. Furthermore, it is noteworthy that Phe 111 appeared to establish vdw interactions with the purine ring of Aza, in agreement with the experimental finding that substitution of Phe 111 by other amino acids causes decreased Aza activity [4]. Finally, the imidazole ring interacts directly with Arg 15 (Figure 6), in line with its general importance for the catalytic function of alpha class GSTs [27,28].…”

“…In a previous study, we integrated the assumed reaction mechanism and molecular docking into a semirational enzyme engineering approach and generated an active-site mutant library of high quality [4]. In the subsequent screening of the focused mutant library for an enzyme with enhanced catalytic efficiency with the prodrug Aza, we found a triple mutant (GDH) of human GST A2-2*E with 70-fold elevated activity over the wild-type [4]. The mutant GST had the following amino acid substitutions in the active site: L107G, L108D and F222H.…”

“…Functional promiscuity is particularly prominent in detoxication enzymes such as GSTs (glutathione transferases), which are involved in the biotransformation and inactivation of innumerable mutagens and other electrophilic compounds [3]. In the present study we have investigated the alternative trajectories from the wild-type human GST A2-2 leading to a triple-point mutant with enhanced catalytic efficiency with Aza (azathioprine) [4] by monitoring the activities with eight alternative substrates. Thermostability and expressivity of the enzyme protein that are other variables of significance to evolution [5][6][7][8] were further investigated.…”

Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

“…However, the conditions under which they are used often differ drastically from their natural environment, which decreases their catalytic performance and restricts their application (2). Consequently, increasing attention is being paid to improving the catalytic performance of enzymes under extreme but application-relevant conditions such as high temperature, strong acid and alkali, and oxidative stress (3)(4)(5).…”

“…The commonly used protein engineering strategies include site-directed mutagenesis and directed evolution (e.g., errorprone PCR) (4)(5)(6)(7)(8)(9)(10)(11). However, there are some disadvantages for their applications for improving the catalytic efficiency.…”