Protein engineering can be used to tailor enzymes for medical purposes, including antibody-directed enzyme prodrug therapy (ADEPT), which can act as a tumor-targeted alternative to conventional chemotherapy for cancer. In ADEPT, the antibody serves as a vector, delivering a drug-activating enzyme selectively to the tumor site. Glutathione transferases (GSTs) are a family of naturally occurring detoxication enzymes, and the finding that some of them are overexpressed in tumors has been exploited to develop GST-activated prodrugs. The prodrug Telcyta is activated by GST P1-1, which is the GST most commonly elevated in cancer cells, implying that tumors overexpressing GST P1-1 should be particularly vulnerable to Telcyta. Promising antitumor activity has been noted in clinical trials, but the wildtype enzyme has modest activity with Telcyta, and further functional improvement would enhance its usefulness for ADEPT. We utilized protein engineering to construct human GST P1-1 gene variants in the search for enzymes with enhanced activity with Telcyta. The variant Y109H displayed a 2.9-fold higher enzyme activity compared to the wild-type GST P1-1. However, increased catalytic potency was accompanied by decreased thermal stability of the Y109H enzyme, losing 99% of its activity in 8 min at 50 °C. Thermal stability was restored by four additional mutations simultaneously introduced without loss of the enhanced activity with Telcyta. The mutation Q85R was identified as an important contributor to the regained thermostability. These results represent a first step towards a functional ADEPT application for Telcyta.

Glutathione transferases are detoxication enzymes with broad catalytic diversity, and small alterations to the protein’s primary structure can have considerable effects on the enzyme’s substrate selectivity profile. We demonstrate that two point mutations in glutathione transferase P1-1 suffice to generate 20-fold enhanced non-selenium-dependent peroxidase activity indicating a facile evolutionary trajectory. Designed mutant libraries of the enzyme were screened for catalytic activities with alternative substrates representing four divergent chemistries. The chemical reactions comprised aromatic substitution, Michael addition, thiocarbamoylation, and hydroperoxide reduction. Two mutants, R1 (Y109H) and an R1-based mutant V2 (Q40M-E41Q-A46S-Y109H-V200L), were discovered with 16.3- and 30-foldincreased peroxidase activity with cumene hydroperoxide (CuOOH) compared to the wildtype enzyme, respectively. The basis of the improved peroxidase activity of the mutant V2 was elucidated by constructing double-point mutants. The mutants V501 (Q40M-Y109H) and V503 (E41Q-Y109H) were found to have 20- and 21-fold improvements in peroxidase activity relative to the wildtype enzyme, respectively. The steady-state kinetic profiles of mutants R1 and V2 in the reduction of CuOOH were compared to the wildtype parameters. The k_cat values for R1 and V2 were 34- and 57-fold higher, respectively, than that of the wildtype enzyme, whereas the mutant K_m values were increased approximately 3-fold. A 10-fold increased catalytic efficiency (k_cat/K_m) in CuOOH reduction is accomplished by the Tyr109His point mutation in R1. The 23-fold increase of the efficiency obtained in V2 was caused by adding further mutations primarily enhancing k_cat. In all mutants with elevated peroxidase activity, His109 played a pivotal role.

The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 μmol min⁻¹ mg⁻¹. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.

Investigations of the effect of antioxidants on idiopathic Parkinson’s disease have been unsuccessful because the preclinical models used to propose these clinical studies do not accurately represent the neurodegenerative process of the disease. Treatment with certain exogenous neurotoxins induces massive and extremely rapid degeneration; for example, MPTP causes severe Parkinsonism in just three days, while the degenerative process of idiopathic Parkinson´s disease proceeds over many years. The endogenous neurotoxin aminochrome seems to be a good alternative target since it is formed in the nigrostriatal system neurons where the degenerative process occurs. Aminochrome induces all the mechanisms reported to be involved in the degenerative processes of idiopathic Parkinson’s disease. The presence of neuromelanin-containing dopaminergic neurons in the postmortem brain of healthy elderly people suggests that neuromelanin synthesis is a normal and harmless process despite the fact that it requires oxidation of dopamine to three ortho-quinones that are potentially toxic, especially aminochrome. The apparent contradiction that neuromelanin synthesis is harmless, despite its formation via neurotoxic ortho-quinones, can be explained by the protective roles of DT-diaphorase and glutathione transferase GSTM2-2 as well as the neuroprotective role of astrocytes secreting exosomes loaded with GSTM2-2. Increasing the expression of DT-diaphorase and GSTM2-2 may be a therapeutic goal to prevent the degeneration of new neuromelanin-containing dopaminergic neurons. Several phytochemicals that induce DT-diaphorase have been discovered and, therefore, an interesting question is whether these phytochemical KEAP1/NRF2 activators can inhibit or decrease aminochrome-induced neurotoxicity.

The glutathione transferase A3-3 (GST A3-3) homodimeric enzyme is the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not been verified in other mammalian species, even though pig and cattle homologs have been cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene duplications (e.g., 12 in the human genome); consequently, the GSTA3 gene is not annotated in most genomes. To improve our understanding of GSTA3 gene products and their functions across diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs from the testes of a dog, goat, and gray short-tailed opossum, the genomes of which all currently lack GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein sequences had a high level of conservation with human GSTA3 mRNA and protein sequences (≥70% and ≥64% identities, respectively). Sequence conservation was also apparent for the 12 residues of the “H-site” in the 222 amino acid GSTA3 protein that is known to interact with the steroid substrates. Modeling predicted that the dog GSTA3-3 may be a more active ketosteroid isomerase than the corresponding goat or opossum enzymes. However, expression of the GSTA3 gene was higher in liver than in other dog tissue. Our results improve understanding of the active sites of mammalian GST A3-3 enzymes, inhibitors of which might be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases.

Novel means of drug delivery are in great demand in particular for the administration of macromolecules. Glutathione transferase proteins can undergo cellular uptake and thereby deliver pharmaceutical cargo to intracellular sites. The proteins are soluble and can be produced in large quantities by facile bacterial expression. Molecular engineering may endow the proteins with signal sequences directing the delivery to specific intracellular targets and escape domains that facilitate cargo release from endosomes. Sequences providing high affinity for nucleic acids may find use in vaccines based on DNA or RNA. The combination of chitosan and glutathione transferase synergistically enhances the cellular uptake of the protein. Engineered glutathione transferase proteins offer great potential in the area of drug delivery research.

Nobo is a glutathione transferase (GST) crucially contributing to ecdysteroid biosynthesis in insects of the orders Diptera and Lepidoptera. Ecdysone is a vital steroid hormone in insects, which governs larval molting and metamorphosis, and the suppression of its synthesis has potential as a novel approach to insect growth regulation and combatting vectors of disease. In general, GSTs catalyze detoxication, whereas the specific function of Nobo in ecdysteroidogenesis is unknown. We report that Nobo from the malaria-spreading mosquito Anopheles gambiae is a highly efficient ketosteroid isomerase catalyzing double-bond isomerization in the steroids 5-androsten-3,17-dione and 5-pregnen-3,20-dione. These mammalian ketosteroids are unknown in mosquitoes, but the discovered prominent catalytic activity of these compounds suggests that the unknown Nobo substrate in insects has a ketosteroid functionality. Aminoacid residue Asp111 in Nobo is essential for activity with the steroids, but not for conventional GST substrates. Further characterization of Nobo may guide the development of new insecticides to prevent malaria.

For more than 60 years, glutathione transferases (GSTs) have attracted attention, but the research field of the GSTome [1] has not yet matured. Originally discovered in the research on cellular protection against carcinogens and mutagens [2], GSTs were, by contrast, in cancer cells found to protect tumors against anti-cancer drugs. The biotransformation of other drugs was also established, in many cases following metabolic activation by cytochrome P450 enzymes. It was further concluded that a crucial GST function was the inactivation of genotoxic electrophiles such as unsaturated aldehydes formed in endogenous processes [3]. The engineering of GSTs for biotechnical applications followed suit [4]. Since then, new aspects have emerged every few years, and a full understanding of the scope of the GST proteins is not yet in sight.

Astrocytes protect neurons by modulating neuronal function and survival. Astrocytes support neurons in several ways. They provide energy through the astrocyte-neuron lactate shuttle, protect neurons from excitotoxicity, and internalize neuronal lipid droplets to degrade fatty acids for neuronal metabolic and synaptic support, as well as by their high capacity for glutamate uptake and the conversion of glutamate to glutamine. A recent reported astrocyte system for protection of dopamine neurons against the neurotoxic products of dopamine, such as aminochrome and other o-quinones, were generated under neuromelanin synthesis by oxidizing dopamine catechol structure. Astrocytes secrete glutathione transferase M2-2 through exosomes that transport this enzyme into dopaminergic neurons to protect these neurons against aminochrome neurotoxicity. The role of this new astrocyte protective mechanism in Parkinson´s disease is discussed.

Glutathione is an important antioxidant that plays a crucial role in the cellular protection against oxidative stress and detoxification of electrophilic mutagens, and carcinogens. Glutathione transferases are enzymes catalyzing glutathione-dependent reactions that lead to inactivation and conjugation of toxic compounds, processes followed by subsequent excretion of the detoxified products. Degeneration and loss of neuromelanin-containing dopaminergic neurons in the nigrostriatal neurons generally involves oxidative stress, neuroinflammation, alpha-synuclein aggregation to neurotoxic oligomers, mitochondrial dysfunction, protein degradation dysfunction, and endoplasmic reticulum stress. However, it is still unclear what triggers these neurodegenerative processes. It has been reported that aminochrome may elicit all of these mechanisms and, interestingly, aminochrome is formed inside neuromelanin-containing dopaminergic neurons during neuromelanin synthesis. Aminochrome is a neurotoxic ortho-quinone formed in neuromelanin synthesis. However, it seems paradoxical that the neurotoxin aminochrome is generated during neuromelanin synthesis, even though healthy seniors have these neurons intact when they die. The explanation of this paradox is the existence of protective tools against aminochrome neurotoxicity composed of the enzymes DT-diaphorase, expressed in these neurons, and glutathione transferase M2-2, expressed in astrocytes. Recently, it has been reported that dopaminergic neurons can be protected by glutathione transferase M2-2 from astrocytes, which secrete exosomes containing the protective enzyme.