Gamma-butyrobetaine dioxygenase

Gamma-butyrobetaine dioxygenase (also known as BBOX, GBBH or γ-butyrobetaine hydroxylase) is an is_associated_with::enzyme that in humans is encoded by the BBOX1 is_associated_with::gene. Gamma-butyrobetaine dioxygenase catalyses the formation of is_associated_with::L-carnitine from gamma-butyrobetaine, the last step in the L-is_associated_with::carnitine biosynthesis pathway. is_associated_with::Carnitine is essential for the transport of activated is_associated_with::fatty acids across the is_associated_with::mitochondrial membrane during mitochondrial is_associated_with::beta oxidation. In humans, gamma-butyrobetaine dioxygenase can be found in kidney (high), liver (moderate), and brain (very low). BBOX1 has recently been identified as a potential is_associated_with::cancer is_associated_with::gene on the basis of a large-scale is_associated_with::microarray data analysis.

Reaction
The following reaction is catalyzed by gamma-butyrobetaine dioxygenase:
 * 4-trimethylammoniobutanoate (γ-butyrobetaine) + is_associated_with::2-oxoglutarate + O2 $$\rightleftharpoons$$ 3-hydroxy-4-trimethylammoniobutanoate (is_associated_with::L-carnitine) + is_associated_with::succinate + CO2

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

This enzyme belongs to the family of is_associated_with::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 is_associated_with::lysine degradation. is_associated_with::Iron is a cofactor for gamma-butyrobetaine dioxygenase. Similar to many other 2OG is_associated_with::oxygenases, the activity of gamma-butyrobetaine dioxygenase can be stimulated by is_associated_with::reducing agents such as is_associated_with::ascorbate and is_associated_with::glutathione. The catalytic activity of gamma-butyrobetaine dioxygenase can be stimulated with different metal ions, especially potassium ions.

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

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

Inhibition
Gamma-butyrobetaine dioxygenase is an inhibition target for 3-(2,2,2-trimethylhydraziniumyl)propionate (is_associated_with::mildronate, also known as THP, MET-88, Meldonium or Quarterine). Mildronate is clinically used for the treatments of is_associated_with::angina and is_associated_with::myocardial infarction. Some studies suggested that mildronate may also be beneficial for the treatment of is_associated_with::neurological disorder, is_associated_with::diabetes, and is_associated_with::seizures and is_associated_with::alcohol intoxication. Mildronate is currently manufactured and marketed by is_associated_with::Grindeks, a pharmaceutical company based in is_associated_with::Latvia. To date, at least five is_associated_with::clinical trial reports were published in peer-reviewed journals documenting the is_associated_with::efficacy and safety of mildronate on the treatments of is_associated_with::angina, is_associated_with::stroke and is_associated_with::chronic heart failure.

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). To date, most is_associated_with::enzyme inhibitors for human 2OG is_associated_with::oxygenases bind to the cosubstrate 2OG is_associated_with::binding site; mildronate is a rare example of a non-peptidyl substrate mimic inhibitor. Although initial reports suggested mildronate is a is_associated_with::non-competitive and non-hydroxylatable analogue of gamma-butyrobetaine, further studies have identified mildronate is indeed a substrate for gamma-butyrobetaine dioxygenase.

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

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