Gamma-butyrobetaine dioxygenase

Gamma-butyrobetaine dioxygenase belongs to the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily. It catalyses the following reaction:

4-trimethylammoniobutanoate (γ-butyrobetaine) + 2-oxoglutarate + O₂ ${\displaystyle \rightleftharpoons }$ 3-hydroxy-4-trimethylammoniobutanoate (L-carnitine) + succinate + CO₂

The three substrates of this enzyme are 4-trimethylammoniobutanoate (γ-butyrobetaine), 2-oxoglutarate, and O₂,[10] whereas its three products are 3-hydroxy-4-trimethylammoniobutanoate (L-carnitine), succinate, and carbon dioxide.

This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O₂ as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O₂ with 2-oxoglutarate as one donor, and incorporation of one atom of oxygen into each donor. This enzyme participates in lysine degradation. Iron is a cofactor for gamma-butyrobetaine dioxygenase. Similar to many other 2OG oxygenases, the activity of gamma-butyrobetaine dioxygenase can be stimulated by reducing agents such as ascorbate and glutathione.[11][12][13][14] The catalytic activity of gamma-butyrobetaine dioxygenase can be stimulated with different metal ions, especially potassium ions.[15]

Both the apo (PDB id: 3N6W)[16] and the holo (PDB id: 3O2G)[17] structures of gamma-butyrobetaine dioxygenase have been solved, demonstrating an induced fit mechanism may contribute to the catalytic activity of gamma-butyrobetaine dioxygenase.

Gamma-butyrobetaine dioxygenase is promiscuous in substrate selectivity and it processes a number of modified substrates, including the natural catalytic products L-carnitine and D-carnitine, forming 3-dehydrocarnitine and trimethylaminoacetone.[17][18] Gamma-butyrobetaine dioxygenase also catalyses the oxidation of mildronate[19] to form multiple products including malonic acid semialdehyde, dimethylamine, formaldehyde and (1-methylimidazolidin-4-yl)acetic acid, which is proposed to be formed via a Stevens rearrangement mechanism.[20][21] Gamma-butyrobetaine dioxygenase is unique among other human 2OG oxygenases that it catalyses both hydroxylation (e.g.: L-carnitine), demethylation (e.g.: formaldehyde) and C-C bond formation (e.g.: (1-methylimidazolidin-4-yl)acetic acid).[22]

Gamma-butyrobetaine dioxygenase is an inhibition target for 3-(2,2,2-trimethylhydraziniumyl)propionate (mildronate, also known as THP, MET-88, Meldonium or Quarterine). Mildronate is offered, clinically, to non-U.S. markets, in treatment of angina and myocardial infarction.[23][24][25] Some studies suggested that mildronate may also be beneficial for the treatment of neurological disorder,[26][27] diabetes,[28] and seizures and alcohol intoxication.[29] Mildronate is currently manufactured and marketed by Grindeks, a pharmaceutical company based in Latvia. To date, at least five clinical trial reports were published in peer-reviewed journals documenting the efficacy and safety of mildronate on the treatments of angina, stroke and chronic heart failure.[30][31][32][33][34] However, there have been no randomized clinical trials to support the use of mildronate to treat any cardiovascular disease.[35]

Mildronate has a similar structure to the natural substrate gamma-butyrobetaine, with a NH group replacing the CH₂ of gamma-butyrobetaine at the C-4 position. A crystal structure of mldronate in complex with gamma-butyrobetaine dioxygenase was published, and it suggests mildronate bind to gamma-butyrobetaine dioxygenase in exactly the same way as gamma-butyrobetaine (PDB id: 3MS5).[36] To date, most enzyme inhibitors for human 2OG oxygenases bind to the cosubstrate 2OG binding site; mildronate is a rare example of a non-peptidyl substrate mimic inhibitor.[37] Although initial reports suggested mildronate is a non-competitive and non-hydroxylatable analogue of gamma-butyrobetaine,[38] further studies have identified mildronate is indeed a substrate for gamma-butyrobetaine dioxygenase.[17][20][39]

Similar to other 2OG oxygenases, gamma-butyrobetaine dioxygenase can be inhibited by 2OG mimics and aromatic inhibitors such as pyridine 2,4-dicarboxylate.[40] Other reported gamma-butyrobetaine dioxygenase inhibitors include cyclopropyl-substituted gamma-butyrobetaines[41] and 3-(2,2-dimethylcyclopropyl)propanoic acid, which is a mechanism-based enzyme inhibitor.[42]

Several in vitro biochemical assays have been applied to monitor the catalytic activity of gamma-butyrobetaine dioxygenase. Early methods have mainly focused on the use of radiolabeled compounds, including ¹⁴C-labelled gamma-butyrobetaine[43] and ¹⁴C-labelled 2OG.[44] Enzyme-coupled method have also been applied to detect carnitine formation, by using the enzyme carnitine acetyltransferase and ¹⁴C-labelled acetyl-coenzyme A to give labelled acetylcarnitine for detection. Using this method, it is possible to detect carnitine concentration down to the pico-molar range.[45][46][47] Other analytical methods including mass spectrometry and NMR have also been applied,[17] and they are in particularly useful for the study of the coupling ratio between 2OG oxidation and substrate formation, and for the characterisation of unknown enzymatic products.[18] However, these methods are often not suitable for high-throughput screening and require expensive instrumentation. A potentially high-throughput fluorescence-based assay has also been proposed by using a fluorinated-gamma-butyrobetaine analog.[48] The fluoride ions released as a result of gamma-butyrobetaine dioxygenase catalyses can be detected by using chemosensors such as protected fluorescein.[49]

• Carnitine
• Mildronate
• Carnitine biosynthesis
• Beta Oxidation
• Fatty acid metabolism
• Stevens rearrangement