Gamma-butyrobetaine dioxygenase explained

Gamma-butyrobetaine dioxygenase (also known as BBOX, GBBH or γ-butyrobetaine hydroxylase) is an enzyme that in humans is encoded by the BBOX1 gene.[1] [2] Gamma-butyrobetaine dioxygenase catalyses the formation of L-carnitine from gamma-butyrobetaine, the last step in the L-carnitine biosynthesis pathway.[3] Carnitine is essential for the transport of activated fatty acids across the mitochondrial membrane during mitochondrial beta oxidation. In humans, gamma-butyrobetaine dioxygenase can be found in the kidney (high), liver (moderate), and brain (very low).[1] [4] BBOX1 has recently been identified as a potential cancer gene based on a large-scale microarray data analysis.[5]

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gamma-butyrobetaine dioxygenase
Ec Number:1.14.11.1
Cas Number:9045-31-2
Go Code:0008336

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

4-trimethylammoniobutanoate (γ-butyrobetaine) + 2-oxoglutarate + O2

\rightleftharpoons

3-hydroxy-4-trimethylammoniobutanoate (L-carnitine) + succinate + CO2

The three substrates of this enzyme are 4-trimethylammoniobutanoate (γ-butyrobetaine), 2-oxoglutarate, and O2,[6] 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.[7] [8] [9] [10] The catalytic activity of gamma-butyrobetaine dioxygenase can be stimulated with different metal ions, especially potassium ions.[11]

Both the apo (PDB id: 3N6W)[12] and the holo (PDB id: 3O2G)[13] 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.[13] [14] Gamma-butyrobetaine dioxygenase also catalyses the oxidation of mildronate[15] 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.[16] [17] 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).[18]

Inhibition

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.[19] [20] [21] Some studies suggested that mildronate may also be beneficial for the treatment of neurological disorder,[22] [23] diabetes,[24] and seizures and alcohol intoxication.[25] 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.[26] [27] [28] [29] [30] However, there have been no randomized clinical trials to support the use of mildronate to treat any cardiovascular disease.[31]

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 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.[32] Although initial reports suggested mildronate is a non-competitive and non-hydroxylatable analogue of gamma-butyrobetaine,[33] further studies have identified mildronate is indeed a substrate for gamma-butyrobetaine dioxygenase.[13] [16] [34]

Similar to other 2OG oxygenases, gamma-butyrobetaine dioxygenase can be inhibited by 2OG mimics and aromatic inhibitors such as pyridine 2,4-dicarboxylate.[35] Other reported gamma-butyrobetaine dioxygenase inhibitors include cyclopropyl-substituted gamma-butyrobetaines[36] and 3-(2,2-dimethylcyclopropyl)propanoic acid, which is a mechanism-based enzyme inhibitor.[37]

Assay

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[38] and 14C-labelled 2OG.[39] 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.[40] [41] [42] Other analytical methods including mass spectrometry and NMR have also been applied,[13] 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.[14] 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.[43] The fluoride ions released as a result of gamma-butyrobetaine dioxygenase catalyses can be detected by using chemosensors such as protected fluorescein.[44]

See also

Further reading

Notes and References

  1. Vaz FM, van Gool S, Ofman R, Ijlst L, Wanders RJ . Carnitine biosynthesis: identification of the cDNA encoding human gamma-butyrobetaine hydroxylase . Biochemical and Biophysical Research Communications . 250 . 2 . 506–10 . Sep 1998 . 9753662 . 10.1006/bbrc.1998.9343 .
  2. Web site: Entrez Gene: BBOX1 butyrobetaine (gamma), 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1.
  3. Paul HS, Sekas G, Adibi SA . Carnitine biosynthesis in hepatic peroxisomes. Demonstration of gamma-butyrobetaine hydroxylase activity . European Journal of Biochemistry . 203 . 3 . 599–605 . Feb 1992 . 1735445 . 10.1111/j.1432-1033.1992.tb16589.x . free .
  4. Lindstedt G, Lindstedt S, Nordin I . Gamma-butyrobetaine hydroxylase in human kidney . Scandinavian Journal of Clinical and Laboratory Investigation . 42 . 6 . 477–85 . Oct 1982 . 7156861 . 10.3109/00365518209168117 .
  5. Dawany NB, Dampier WN, Tozeren A . Large-scale integration of microarray data reveals genes and pathways common to multiple cancer types . International Journal of Cancer . 128 . 12 . 2881–91 . Jun 2011 . 21165954 . 10.1002/ijc.25854 . 24740881 . free .
  6. Lindstedt G, Lindstedt S . Cofactor requirements of gamma-butyrobetaine hydroxylase from rat liver . The Journal of Biological Chemistry . 245 . 16 . 4178–86 . Aug 1970 . 10.1016/S0021-9258(18)62901-1 . 4396068 . free .
  7. Rebouche CJ . Ascorbic acid and carnitine biosynthesis . The American Journal of Clinical Nutrition . 54 . 6 Suppl . 1147S–1152S . Dec 1991 . 1962562 . 10.1093/ajcn/54.6.1147s. free .
  8. Nelson PJ, Pruitt RE, Henderson LL, Jenness R, Henderson LM . Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine . Biochimica et Biophysica Acta (BBA) - General Subjects . 672 . 1 . 123–7 . Jan 1981 . 6783120 . 10.1016/0304-4165(81)90286-5 .
  9. Rebouche CJ . Ascorbic acid and carnitine biosynthesis . The American Journal of Clinical Nutrition . 54 . 6 Suppl . 1147S–1152S . Dec 1991 . 1962562 . 10.1093/ajcn/54.6.1147s . free .
  10. Furusawa H, Sato Y, Tanaka Y, Inai Y, Amano A, Iwama M, Kondo Y, Handa S, Murata A, Nishikimi M, Goto S, Maruyama N, Takahashi R, Ishigami A . Vitamin C is not essential for carnitine biosynthesis in vivo: verification in vitamin C-depleted senescence marker protein-30/gluconolactonase knockout mice . Biological & Pharmaceutical Bulletin . 31 . 9 . 1673–9 . Sep 2008 . 18758058 . 10.1248/bpb.31.1673 . free .
  11. Wehbie RS, Punekar NS, Lardy HA . Rat liver gamma-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogues on hydroxylation and decarboxylation . Biochemistry . 27 . 6 . 2222–8 . Mar 1988 . 3378057 . 10.1021/bi00406a062 .
  12. Tars K, Rumnieks J, Zeltins A, Kazaks A, Kotelovica S, Leonciks A, Sharipo J, Viksna A, Kuka J, Liepinsh E, Dambrova M . Crystal structure of human gamma-butyrobetaine hydroxylase . Biochemical and Biophysical Research Communications . 398 . 4 . 634–9 . Aug 2010 . 20599753 . 10.1016/j.bbrc.2010.06.121 .
  13. Leung IK, Krojer TJ, Kochan GT, Henry L, von Delft F, Claridge TD, Oppermann U, McDonough MA, Schofield CJ . Structural and mechanistic studies on γ-butyrobetaine hydroxylase . Chemistry & Biology . 17 . 12 . 1316–24 . Dec 2010 . 21168767 . 10.1016/j.chembiol.2010.09.016 . free .
  14. Fujimori DG . A novel enzymatic rearrangement . Chemistry & Biology . 17 . 12 . 1269–70 . Dec 2010 . 21168760 . 10.1016/j.chembiol.2010.12.003 . free .
  15. Simkhovich BZ, Shutenko ZV, Meirena DV, Khagi KB, Mezapuķe RJ, Molodchina TN, Kalviņs IJ, Lukevics E . 3-(2,2,2-Trimethylhydrazinium)propionate (THP)--a novel gamma-butyrobetaine hydroxylase inhibitor with cardioprotective properties . Biochemical Pharmacology . 37 . 2 . 195–202 . Jan 1988 . 3342076 . 10.1016/0006-2952(88)90717-4 .
  16. Henry L, Leung IK, Claridge TD, Schofield CJ . γ-Butyrobetaine hydroxylase catalyses a Stevens type rearrangement . Bioorganic & Medicinal Chemistry Letters . 22 . 15 . 4975–8 . Aug 2012 . 22765904 . 10.1016/j.bmcl.2012.06.024 .
  17. Stevens TS, Creighton EM, Gordon AB, MacNicol M . 1928 . CCCCXXIII.—Degradation of quaternary ammonium salts. Part I . J. Chem. Soc. . 3193 - 3197 . 10.1039/JR9280003193 .
  18. Loenarz C, Schofield CJ . Expanding chemical biology of 2-oxoglutarate oxygenases . Nature Chemical Biology . 4 . 3 . 152–6 . Mar 2008 . 18277970 . 10.1038/nchembio0308-152 .
  19. Sesti C, Simkhovich BZ, Kalvinsh I, Kloner RA . Mildronate, a novel fatty acid oxidation inhibitor and antianginal agent, reduces myocardial infarct size without affecting hemodynamics . Journal of Cardiovascular Pharmacology . 47 . 3 . 493–9 . Mar 2006 . 16633095 . 10.1097/01.fjc.0000211732.76668.d2 . 45844835 . free .
  20. Liepinsh E, Vilskersts R, Loca D, Kirjanova O, Pugovichs O, Kalvinsh I, Dambrova M . Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction . Journal of Cardiovascular Pharmacology . 48 . 6 . 314–9 . Dec 2006 . 17204911 . 10.1097/01.fjc.0000250077.07702.23 . 1812127 . free .
  21. Hayashi Y, Kirimoto T, Asaka N, Nakano M, Tajima K, Miyake H, Matsuura N . Beneficial effects of MET-88, a gamma-butyrobetaine hydroxylase inhibitor in rats with heart failure following myocardial infarction . European Journal of Pharmacology . 395 . 3 . 217–24 . May 2000 . 10812052 . 10.1016/S0014-2999(00)00098-4 .
  22. Sjakste N, Gutcaits A, Kalvinsh I . Mildronate: an antiischemic drug for neurological indications . CNS Drug Reviews . 11 . 2 . 151–68 . 2005 . 16007237 . 6741751 . 10.1111/j.1527-3458.2005.tb00267.x .
  23. Pupure J, Isajevs S, Skapare E, Rumaks J, Svirskis S, Svirina D, Kalvinsh I, Klusa V . Neuroprotective properties of mildronate, a mitochondria-targeted small molecule . Neuroscience Letters . 470 . 2 . 100–5 . Feb 2010 . 20036318 . 10.1016/j.neulet.2009.12.055 . 38603504 .
  24. Liepinsh E, Skapare E, Svalbe B, Makrecka M, Cirule H, Dambrova M . Anti-diabetic effects of mildronate alone or in combination with metformin in obese Zucker rats . European Journal of Pharmacology . 658 . 2–3 . 277–83 . May 2011 . 21371472 . 10.1016/j.ejphar.2011.02.019 .
  25. Zvejniece L, Svalbe B, Makrecka M, Liepinsh E, Kalvinsh I, Dambrova M . Mildronate exerts acute anticonvulsant and antihypnotic effects . Behavioural Pharmacology . 21 . 5–6 . 548–55 . Sep 2010 . 20661137 . 10.1097/FBP.0b013e32833d5a59 . 12501700 .
  26. Dzerve V, Matisone D, Kukulis I, Romanova J, Putane L, Grabauskiene V, Skarda I, Berzina D, Strautmanis J . Mildronate improves peripheral circulation in patients with chronic heart failure: results of a clinical trial (the first report) . Semin Cardiol . 11 . 2 . 56–64 . 2005 . 1648-7966 . https://web.archive.org/web/20120328094306/http://www.seminarsincardiology.com/pdf/Seminars-2005-11-2-56-64.pdf . dead . 2012-03-28 .
  27. Vitols A, Voita D, Dzerve V . Mildronate improves carotid baroreceptor reflex function in patients with chronic heart failure . Semin Cardiovasc Med . 13 . 6 . 2008 . https://web.archive.org/web/20130921060004/http://www.seminarsincardiology.com/pdf/SeminarsCVMed-2007-13-6.pdf . dead . 2013-09-21 .
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  31. News: Tennis pro Maria Sharapova says she takes 'full responsibility' for failed drug test . LA Times . 8 March 2016 .
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  34. Spaniol M, Brooks H, Auer L, Zimmermann A, Solioz M, Stieger B, Krähenbühl S . Development and characterization of an animal model of carnitine deficiency . European Journal of Biochemistry . 268 . 6 . 1876–87 . Mar 2001 . 11248709 . 10.1046/j.1432-1327.2001.02065.x . free .
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