How Glycerol Boosts Catalytic Efficiency in Thiol Dioxygenase (ADO)

A recent study investigates how substrate binding influences the activity of cysteamine dioxygenase (ADO), one of the only known thiol dioxygenases in mammals. ADO catalyzes the oxygen-dependent oxidation of cysteamine and also regulates protein stability through oxidation of N-terminal cysteine residues.¹ Although substrates are generally thought to bind its iron active site in a bidentate manner, the exact coordination in ADO has been unclear.¹ The researchers demonstrated that molecular crowding by glycerol shifts cysteamine binding toward a bidentate mode, similar to that seen in protein substrates. Spectroscopic analyses and computational modeling show that glycerol stabilizes this binding configuration, leading to a roughly sevenfold increase in catalytic efficiency.¹

To discuss the study’s findings, Spectroscopy sat down with Brad Pierce, a Professor at the University of Alabama,² and Joshua Helms, a graduate student at the University of Alabama, who were the lead authors of this study. In the first part of our two-part interview with Pierce and Helms, they talk about how ADO can be a uniquely dual-function enzyme that links metabolism and oxygen sensing.

Can you talk about ADO's dual functionality and its implications for oxygen sensing and protein turnover?

ADO is an unusual enzyme because it serves two distinct biological functions. First, it acts as a conventional metabolic enzyme, converting molecular oxygen and cysteamine into hypotaurine. This feeds into the biosynthesis of taurine, a molecule important for heart function, hydration balance, and nerve signaling. In addition, ADO also acts as an oxygen sensor by chemically modifying specific eukaryotic proteins at their exposed N-terminal cysteine residues. This post-translational modification marks those proteins for degradation through a cellular pathway referred to as the N-degron pathway. What makes this particularly interesting is that the same chemical reaction, O₂-dependent oxidation of a thiol-bearing functional group, serves both a metabolic and a regulatory function depending on the substrate. Because molecular oxygen is a co-substrate for both ADO-catalyzed reactions, it effectively links the cellular oxygen concentration to decisions about protein stability. When oxygen is available, ADO can degrade regulatory proteins like G-protein signaling regulators. However, when oxygen is scarce, that degradation slows. This positions ADO as a molecular bridge connecting the cellular concentration of molecular oxygen to protein turnover, a connection that has broad implications for how cells adapt to low-oxygen conditions like those found in tumors cells or at high altitude.

What motivated the investigation into bidentate versus monodentate binding, and why has it remained unresolved?

This question really gets at a fundamental issue in enzyme biochemistry. Namely, of what significance is the structure of the enzyme–substrate complex and how does this promote efficient catalytic turnover? The so-called, small molecule thiol dioxygenases (smTDOs) such as cysteine dioxygenase (CDO) and 3-mercaptopropionate dioxygenase (MDO), have been extensively studied. Among these enzymes, the substrate interacts with the iron center through two simultaneous points of contact. This “bidentate” coordination makes the iron site more reactive toward molecular oxygen and imparts high chemo- and stereoselectively to the reaction. ADO, and the closely related plant cysteine oxidase (PCO), catalyze the same chemical reactions as smTDOs but exhibit significant divergence in both amino acid sequence and overall structure.

Earlier studies using different spectroscopic methods reached contradictory conclusions regarding the nature of substrate binding to ADO. Initial studies relying on magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) suggested cysteamine attached to the iron site through the substrate thiol-group only (monodentate). Conversely, a recent X-ray crystallographic study using a cyclic polypeptide inhibitor suggested bidentate coordination was more likely.

As summarized in this report, part of the confusion stemmed from a seemingly mundane experimental variable. We discovered that glycerol, which is routinely employed as a protein stabilizer, cryoprotectant, and spectroscopic glassing agent, engages in direct, non-innocent interactions with the ADO enzyme–substrate complex. This interaction alters the environment near the iron center such that the substrate favors bidentate coordination. As it turns out, differing amounts of glycerol have been used among different research groups, and nobody recognized that this was altering the behavior of the enzyme. These results should serve as a broader cautionary note for the enzymology and spectroscopy community that glycerol cannot be assumed to act as an inert spectator in buffer systems. Although unrelated to glycerol, an additional complication was the false equivalence prior studies assumed when considering substrate binding to the oxidized, catalytically inactive Fe(III)-ADO with binding to the catalytically competent Fe(II)-ADO enzyme; an assumption that we demonstrate here is not justified.

How does molecular crowding from glycerol mechanistically drive the shift to bidentate coordination?

Think of the active site of an enzyme as a small, somewhat flexible pocket. We believe that the substrate, cysteamine, initially binds to the iron center through the thiol sulfur atom, thereby leaving the amino group unattached and able to adopt multiple conformations. Only when the amino and thiol groups are simultaneously attached to the iron is the enzyme capable of reacting with molecular oxygen to generate product. As it turns out, glycerol is perfectly sized to occupy a position within the substrate binding channel that limits the conformational space available for cysteamine when bound to the iron center. By filling this space, glycerol restricts the range of positions available to the bound cysteamine, effectively nudging it into the more compact, bidentate configuration where both the sulfur and the amino group coordinate to the iron simultaneously.

Importantly, this effect is not simply due to changes in solution viscosity. When we substituted other larger additives such as sucrose or PEG-200, no increase in enzymatic activity or bidentate coordination was observed. This suggests that glycerol provides a unique balance of size and molecular interactions, which are “just right” to promote bidentate substrate binding.

What does the ~7-fold increase in catalytic efficiency reveal about denticity and O₂ activation?

Although a nearly 10-fold increase in catalytic efficiency (k_cat/K_M) is itself significant, the more important point is that this value serves as a diagnostic indicator of a more “active” form of the enzyme. In addition, the functional assays performed use an enzyme concentration at least two orders of magnitude lower than required for spectroscopic measurements. Consequently, rates of enzymatic turnover were used to rapidly screen for specific conditions which favor an increased fraction of the catalytically active enzyme-substrate complex.

The systematic increase in ADO activity with increasing glycerol indicates that glycerol either enhances the rate of chemical steps in the reaction or it increases the fraction of “active” enzyme present in solution. Since glycerol increases solvent viscosity, and slows molecular motion, it is unlikely that it increases the rates of chemical steps. We can therefore conclude that glycerol must increase the fraction of “active” ADO present in solution.

Once these conditions were identified, EPR studies were performed to directly observe the increased fraction of bidentate enzyme-substrate complex spectroscopically. These EPR experiments use nitric oxide as a stand-in for molecular oxygen. However, these two molecules behave similarly at the iron center, thereby allowing us to confirm that the oxygen surrogate only binds to the iron center when cysteamine is bound in a bidentate configuration.

Using this two-pronged approach, we were able to correlate spectroscopic observables to a functional “active” form of the enzyme.

What will Part 2 of the interview will cover?

The next part of our conversation with Pierce and Helms will cover the following topics:

• How multiple techniques helped build a detailed, complementary picture of how glycerol alters ADO’s active site.
• What spectroscopic methods revealed about glycerol
• How computational modeling supported experimental findings
• What important physiological questions the findings of the study raise

References

1. Helms, J. R.; Probst, M.; Paris, J.; et al. EPR Spectroscopy Reveals Glycerol-dependent Activation of Cysteamine Dioxygenase (ADO) Enables Bidentate Substrate Coordination. J. Biol. Chem. 2026, 111438. DOI: 10.1016/j.jbc.2026.111438
2. University of Alabama, Brad S. Pierce. UA.edu. Available at: https://chemistry.ua.edu/people/bradley-s-pierce/ (accessed 2026-04-27).