Glutathione Transferases as Efficient Ketosteroid Isomerases

In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M−1s−1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3β-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s−1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.

Ensemble–function relationships to evaluate catalysis in the ketosteroid isomerase oxyanion hole

Following decades of insights from structure–function studies, there is now a need to progress from a static to dynamic view of enzymes. Comparison of prior cryo X-ray structures suggested that deleterious effects from ketosteroid isomerase (KSI) mutants arise from misalignment of the oxyanion hole catalytic residue, Y16. However, multi-conformer models from room temperature X-ray diffraction revealed an ensemble of Y16 conformers indistinguishable from WT for Y32F/Y57F KSI and a distinct, non-native ensemble for Y16 in Y57F KSI. Functional analyses suggested rate effects arise from weakened hydrogen bonding, due to disruption of the Y16/Y57/Y32 hydrogen bond network, and repositioning of the general base. In general, catalytic changes can be deconvoluted into effects on the probability of occupying a state (P-effects) and the reactivity of each state (k-effects). Our results underscore the need for ensemble–function analysis to decipher enzyme function and ultimately manipulate their extraordinary capabilities.

Globally Optimized Molecular Embeddings for Dynamic Reaction Solvate Shell Optimization and Active Site Design

We demonstrate how a full QM/MM derivatization of the recently developed GOCAT model can be utilized in the global optimization of molecular embeddings. To this end, we provide two distinct examples: An $$\text {S}_\text {N}2$$ S N 2 reaction, and one enzymatic example of recent interest, the ketosteroid isomerase. These serve us to highlight the advantages of such an approach and sketch the roadmap for further improvements.

Parallel molecular mechanisms for enzyme temperature adaptation

The mechanisms that underly the adaptation of enzyme activities and stabilities to temperature are fundamental to our understanding of molecular evolution and how enzymes work. Here, we investigate the molecular and evolutionary mechanisms of enzyme temperature adaption, combining deep mechanistic studies with comprehensive sequence analyses of thousands of enzymes. We show that temperature adaptation in ketosteroid isomerase (KSI) arises primarily from one residue change with limited, local epistasis, and we establish the underlying physical mechanisms. This residue change occurs in diverse KSI backgrounds, suggesting parallel adaptation to temperature. We identify residues associated with organismal growth temperature across 1005 diverse bacterial enzyme families, suggesting widespread parallel adaptation to temperature. We assess the residue properties, molecular interactions, and interaction networks that appear to underly temperature adaptation.

Conservation of glutathione S-transferase mRNA and protein sequences similar to human and horse Alpha class GST A3-3 across dog, goat, and opossum species

Recently, the glutathione S-transferase A3-3 (GST A3-3) homodimeric enzyme was identified as 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 (12 in the human genome), so few genomes have a corresponding GSTA3 gene annotated. 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, with those current genomes lacking 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 13 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 is a more active ketosteroid isomerase than the goat or opossum enzymes. Our results help us understand the active sites of mammalian GST A3-3 enzymes, and their inhibitors may be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases.