gamma-butyrobetaine dioxygenase

{{infobox enzyme

| align = left

| Name = gamma-butyrobetaine dioxygenase

| EC_number = 1.14.11.1

| CAS_number = 9045-31-2

| GO_code = 0008336

| image =

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

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

The three substrates of this enzyme are 4-trimethylammoniobutanoate (γ-butyrobetaine), 2-oxoglutarate, and O₂,{{cite journal | vauthors = Lindstedt G, Lindstedt S | title = Cofactor requirements of gamma-butyrobetaine hydroxylase from rat liver | journal = The Journal of Biological Chemistry | volume = 245 | issue = 16 | pages = 4178–86 | date = Aug 1970 | doi = 10.1016/S0021-9258(18)62901-1 | pmid = 4396068 | doi-access = free }} 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.{{cite journal | vauthors = Rebouche CJ | title = Ascorbic acid and carnitine biosynthesis | journal = The American Journal of Clinical Nutrition | volume = 54 | issue = 6 Suppl | pages = 1147S–1152S | date = Dec 1991 | pmid = 1962562 | doi = 10.1093/ajcn/54.6.1147s| doi-access = free }}{{cite journal | vauthors = Nelson PJ, Pruitt RE, Henderson LL, Jenness R, Henderson LM | title = Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine | journal = Biochimica et Biophysica Acta (BBA) - General Subjects | volume = 672 | issue = 1 | pages = 123–7 | date = Jan 1981 | pmid = 6783120 | doi = 10.1016/0304-4165(81)90286-5 }}{{cite journal | vauthors = Rebouche CJ | title = Ascorbic acid and carnitine biosynthesis | journal = The American Journal of Clinical Nutrition | volume = 54 | issue = 6 Suppl | pages = 1147S–1152S | date = Dec 1991 | pmid = 1962562 | url = http://www.ajcn.org/content/54/6/1147S.abstract | doi = 10.1093/ajcn/54.6.1147s | doi-access = free }}{{cite journal | vauthors = 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 | title = Vitamin C is not essential for carnitine biosynthesis in vivo: verification in vitamin C-depleted senescence marker protein-30/gluconolactonase knockout mice | journal = Biological & Pharmaceutical Bulletin | volume = 31 | issue = 9 | pages = 1673–9 | date = Sep 2008 | pmid = 18758058 | doi = 10.1248/bpb.31.1673 | doi-access = free }} The catalytic activity of gamma-butyrobetaine dioxygenase can be stimulated with different metal ions, especially potassium ions.{{cite journal | vauthors = Wehbie RS, Punekar NS, Lardy HA | title = Rat liver gamma-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogues on hydroxylation and decarboxylation | journal = Biochemistry | volume = 27 | issue = 6 | pages = 2222–8 | date = Mar 1988 | pmid = 3378057 | doi = 10.1021/bi00406a062 }}

Both the apo (PDB id: 3N6W){{PDB|3N6W}};{{cite journal | vauthors = Tars K, Rumnieks J, Zeltins A, Kazaks A, Kotelovica S, Leonciks A, Sharipo J, Viksna A, Kuka J, Liepinsh E, Dambrova M | title = Crystal structure of human gamma-butyrobetaine hydroxylase | journal = Biochemical and Biophysical Research Communications | volume = 398 | issue = 4 | pages = 634–9 | date = Aug 2010 | pmid = 20599753 | doi = 10.1016/j.bbrc.2010.06.121 }} and the holo (PDB id: 3O2G){{PDB|3O2G}}; {{cite journal | vauthors = Leung IK, Krojer TJ, Kochan GT, Henry L, von Delft F, Claridge TD, Oppermann U, McDonough MA, Schofield CJ | title = Structural and mechanistic studies on γ-butyrobetaine hydroxylase | journal = Chemistry & Biology | volume = 17 | issue = 12 | pages = 1316–24 | date = Dec 2010 | pmid = 21168767 | doi = 10.1016/j.chembiol.2010.09.016 | doi-access = free }} 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.{{cite journal | vauthors = Fujimori DG | title = A novel enzymatic rearrangement | journal = Chemistry & Biology | volume = 17 | issue = 12 | pages = 1269–70 | date = Dec 2010 | pmid = 21168760 | doi = 10.1016/j.chembiol.2010.12.003 | doi-access = free }} Gamma-butyrobetaine dioxygenase also catalyses the oxidation of mildronate{{cite journal | vauthors = Simkhovich BZ, Shutenko ZV, Meirena DV, Khagi KB, Mezapuķe RJ, Molodchina TN, Kalviņs IJ, Lukevics E | title = 3-(2,2,2-Trimethylhydrazinium)propionate (THP)--a novel gamma-butyrobetaine hydroxylase inhibitor with cardioprotective properties | journal = Biochemical Pharmacology | volume = 37 | issue = 2 | pages = 195–202 | date = Jan 1988 | pmid = 3342076 | doi = 10.1016/0006-2952(88)90717-4 }} 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.{{cite journal | vauthors = Henry L, Leung IK, Claridge TD, Schofield CJ | title = γ-Butyrobetaine hydroxylase catalyses a Stevens type rearrangement | journal = Bioorganic & Medicinal Chemistry Letters | volume = 22 | issue = 15 | pages = 4975–8 | date = Aug 2012 | pmid = 22765904 | doi = 10.1016/j.bmcl.2012.06.024 }}{{cite journal | vauthors = Stevens TS, Creighton EM, Gordon AB, MacNicol M | year = 1928 | title = CCCCXXIII.—Degradation of quaternary ammonium salts. Part I | journal = J. Chem. Soc. | pages = 3193–3197 | doi = 10.1039/JR9280003193 }} 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).{{cite journal | vauthors = Loenarz C, Schofield CJ | title = Expanding chemical biology of 2-oxoglutarate oxygenases | journal = Nature Chemical Biology | volume = 4 | issue = 3 | pages = 152–6 | date = Mar 2008 | pmid = 18277970 | doi = 10.1038/nchembio0308-152 }}

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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.{{cite journal | vauthors = Sesti C, Simkhovich BZ, Kalvinsh I, Kloner RA | title = Mildronate, a novel fatty acid oxidation inhibitor and antianginal agent, reduces myocardial infarct size without affecting hemodynamics | journal = Journal of Cardiovascular Pharmacology | volume = 47 | issue = 3 | pages = 493–9 | date = Mar 2006 | pmid = 16633095 | doi = 10.1097/01.fjc.0000211732.76668.d2 | s2cid = 45844835 | doi-access = free }}{{cite journal | vauthors = Liepinsh E, Vilskersts R, Loca D, Kirjanova O, Pugovichs O, Kalvinsh I, Dambrova M | title = Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction | journal = Journal of Cardiovascular Pharmacology | volume = 48 | issue = 6 | pages = 314–9 | date = Dec 2006 | pmid = 17204911 | doi = 10.1097/01.fjc.0000250077.07702.23 | s2cid = 1812127 | doi-access = free }}{{cite journal | vauthors = Hayashi Y, Kirimoto T, Asaka N, Nakano M, Tajima K, Miyake H, Matsuura N | title = Beneficial effects of MET-88, a gamma-butyrobetaine hydroxylase inhibitor in rats with heart failure following myocardial infarction | journal = European Journal of Pharmacology | volume = 395 | issue = 3 | pages = 217–24 | date = May 2000 | pmid = 10812052 | doi = 10.1016/S0014-2999(00)00098-4 }} Some studies suggested that mildronate may also be beneficial for the treatment of neurological disorder,{{cite journal | vauthors = Sjakste N, Gutcaits A, Kalvinsh I | title = Mildronate: an antiischemic drug for neurological indications | journal = CNS Drug Reviews | volume = 11 | issue = 2 | pages = 151–68 | year = 2005 | pmid = 16007237 | pmc = 6741751 | doi = 10.1111/j.1527-3458.2005.tb00267.x }}{{cite journal | vauthors = Pupure J, Isajevs S, Skapare E, Rumaks J, Svirskis S, Svirina D, Kalvinsh I, Klusa V | title = Neuroprotective properties of mildronate, a mitochondria-targeted small molecule | journal = Neuroscience Letters | volume = 470 | issue = 2 | pages = 100–5 | date = Feb 2010 | pmid = 20036318 | doi = 10.1016/j.neulet.2009.12.055 | s2cid = 38603504 }} diabetes,{{cite journal | vauthors = Liepinsh E, Skapare E, Svalbe B, Makrecka M, Cirule H, Dambrova M | title = Anti-diabetic effects of mildronate alone or in combination with metformin in obese Zucker rats | journal = European Journal of Pharmacology | volume = 658 | issue = 2–3 | pages = 277–83 | date = May 2011 | pmid = 21371472 | doi = 10.1016/j.ejphar.2011.02.019 }} and seizures and alcohol intoxication.{{cite journal | vauthors = Zvejniece L, Svalbe B, Makrecka M, Liepinsh E, Kalvinsh I, Dambrova M | title = Mildronate exerts acute anticonvulsant and antihypnotic effects | journal = Behavioural Pharmacology | volume = 21 | issue = 5–6 | pages = 548–55 | date = Sep 2010 | pmid = 20661137 | doi = 10.1097/FBP.0b013e32833d5a59 | s2cid = 12501700 }} 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.{{cite journal|vauthors=Dzerve V, Matisone D, Kukulis I, Romanova J, Putane L, Grabauskiene V, Skarda I, Berzina D, Strautmanis J |title=Mildronate improves peripheral circulation in patients with chronic heart failure: results of a clinical trial (the first report) |journal=Semin Cardiol |volume=11 |issue=2 |pages=56–64 |year=2005 |issn=1648-7966 |url=http://www.seminarsincardiology.com/pdf/Seminars-2005-11-2-56-64.pdf |archive-url=https://web.archive.org/web/20120328094306/http://www.seminarsincardiology.com/pdf/Seminars-2005-11-2-56-64.pdf |url-status=dead |archive-date=2012-03-28 }}{{cite journal|vauthors=Vitols A, Voita D, Dzerve V |title=Mildronate improves carotid baroreceptor reflex function in patients with chronic heart failure |journal=Semin Cardiovasc Med |volume=13 |pages=6 |year=2008 |url=http://www.seminarsincardiology.com/pdf/SeminarsCVMed-2007-13-6.pdf |archive-url=https://web.archive.org/web/20130921060004/http://www.seminarsincardiology.com/pdf/SeminarsCVMed-2007-13-6.pdf |url-status=dead |archive-date=2013-09-21 }}{{cite journal|vauthors=Dzerve V, Matisone D, Pozdnyakov Y, Oganov R |title=Mildronate improves the exercise tolerance in patients with stable angina: results of a long term clinical trial |journal=Semin Cardiovasc Med |volume=16 |pages=3 |year=2010 |url=http://www.seminarsincardiology.com/pdf/SeminarsCVMed-2010-16-3.pdf |url-status=dead |archive-url=https://web.archive.org/web/20130921060011/http://www.seminarsincardiology.com/pdf/SeminarsCVMed-2010-16-3.pdf |archive-date=2013-09-21 }}{{cite journal|vauthors=Dzerve V |title=A dose-dependent improvement in exercise tolerance in patients with stable angina treated with mildronate: a clinical trial "MILSS I" |journal=Medicina |volume=47 |issue=10 |pages=544–51 |year=2011 |pmid=22186118 |doi= 10.3390/medicina47100078|doi-access=free }}{{cite journal | vauthors = Zhu Y, Zhang G, Zhao J, Li D, Yan X, Liu J, Liu X, Zhao H, Xia J, Zhang X, Li Z, Zhang B, Guo Z, Feng L, Zhang Z, Qu F, Zhao G | title = Efficacy and safety of mildronate for acute ischemic stroke: a randomized, double-blind, active-controlled phase II multicenter trial | journal = Clinical Drug Investigation | volume = 33 | issue = 10 | pages = 755–60 | date = Oct 2013 | pmid = 23949899| doi = 10.1007/s40261-013-0121-x | s2cid = 697191 }} However, there have been no randomized clinical trials to support the use of mildronate to treat any cardiovascular disease.{{cite news | url = http://www.latimes.com/sports/sportsnow/la-sp-sn-maria-sharapova-drug-test-20160307-story.html | title = Tennis pro Maria Sharapova says she takes 'full responsibility' for failed drug test | newspaper = LA Times | date = 8 March 2016 }}{{better source needed|date=March 2016}}

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).{{PDB|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.{{cite journal | vauthors = Rose NR, McDonough MA, King ON, Kawamura A, Schofield CJ | title = Inhibition of 2-oxoglutarate dependent oxygenases | journal = Chemical Society Reviews | volume = 40 | issue = 8 | pages = 4364–97 | date = Aug 2011 | pmid = 21390379 | doi = 10.1039/c0cs00203h }} Although initial reports suggested mildronate is a non-competitive and non-hydroxylatable analogue of gamma-butyrobetaine,{{cite journal | vauthors = Galland S, Le Borgne F, Guyonnet D, Clouet P, Demarquoy J | title = Purification and characterization of the rat liver gamma-butyrobetaine hydroxylase | journal = Molecular and Cellular Biochemistry | volume = 178 | issue = 1–2 | pages = 163–8 | date = Jan 1998 | pmid = 9546596 | doi = 10.1023/A:1006849713407 | s2cid = 23339575 }} further studies have identified mildronate is indeed a substrate for gamma-butyrobetaine dioxygenase.{{cite journal | vauthors = Spaniol M, Brooks H, Auer L, Zimmermann A, Solioz M, Stieger B, Krähenbühl S | title = Development and characterization of an animal model of carnitine deficiency | journal = European Journal of Biochemistry | volume = 268 | issue = 6 | pages = 1876–87 | date = Mar 2001 | pmid = 11248709 | doi = 10.1046/j.1432-1327.2001.02065.x | doi-access = free }}

Similar to other 2OG oxygenases, gamma-butyrobetaine dioxygenase can be inhibited by 2OG mimics and aromatic inhibitors such as pyridine 2,4-dicarboxylate.{{cite journal | vauthors = Ng SF, Hanauske-Abel HM, Englard S | title = Cosubstrate binding site of Pseudomonas sp. AK1 gamma-butyrobetaine hydroxylase. Interactions with structural analogs of alpha-ketoglutarate | journal = The Journal of Biological Chemistry | volume = 266 | issue = 3 | pages = 1526–33 | date = Jan 1991 | doi = 10.1016/S0021-9258(18)52326-7 | pmid = 1988434 | doi-access = free }} Other reported gamma-butyrobetaine dioxygenase inhibitors include cyclopropyl-substituted gamma-butyrobetaines{{cite journal | vauthors = Petter RC, Banerjee S, Englard S | year = 1990 | title = Inhibition of γ-butyrobetaine hydroxylase by cyclopropyl-substituted γ-butyrobetaines | journal = J. Org. Chem. | volume = 55 | pages = 3088–3097 | doi = 10.1021/jo00297a025 | issue = 10}} and 3-(2,2-dimethylcyclopropyl)propanoic acid, which is a mechanism-based enzyme inhibitor.{{cite journal | vauthors = Ziering DL, Pascal Jr RA | year = 1990 | title = Mechanism-based inhibition of bacterial γ-butyrobetaine hydroxylase | journal = J. Am. Chem. Soc. | volume = 112 | pages = 834–838 | doi = 10.1021/ja00158a051 | issue = 2 }}

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{{cite journal | vauthors = Lindstedt G, Lindstedt S, Tofft S | year = 1970 | title = γ-Butyrobetaine Hydroxylase from Pseudomonas sp AK 1 | journal = Biochemistry | volume = 9 | pages = 4336–4342 | doi = 10.1021/bi00824a014 | issue = 22 | pmid = 5472709 }} and ¹⁴C-labelled 2OG.{{cite journal | vauthors = Lindstedt G, Lindstedt S, Nordin I | year = 1977 | title = Purification and properties of γ-butyrobetaine hydroxylase from Pseudomonas species AK 1 | journal = Biochemistry | volume = 16 | pages = 2181–2188 | doi = 10.1021/bi00629a022 | issue = 10 | pmid = 861203 }} 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.{{cite journal | vauthors = Cederblad C, Lindstedt S | year = 1972 | title = A method for the determination of carnitine in the picomole range | journal = Clin. Chim. Acta | volume = 37 | pages = 235–243 | doi = 10.1016/0009-8981(72)90438-X | pmid = 5022087 }}{{cite journal | vauthors = Böhmer T, Rydning A, Solberg HE | year = 1974 | title = Carnitine levels in human serum in health and disease | journal = Clin. Chim. Acta | volume = 57 | pages = 55–61 | doi = 10.1016/0009-8981(74)90177-6 | issue = 1 | pmid = 4279150 }}{{cite journal | vauthors = Parvin R, Pande SV | year = 1976 | title = Microdetermination of (−)carnitine and carnitine acetyltransferase activity | journal = Anal. Biochem. | volume = 79 | pages = 190–201 | doi = 10.1016/0003-2697(77)90393-1 | issue = 1–2 | pmid = 869176 }} Other analytical methods including mass spectrometry and NMR have also been applied, 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. 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.{{cite journal | vauthors = Rydzik AM, Leung IK, Kochan GT, Thalhammer A, Oppermann U, Claridge TD, Schofield CJ | title = Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase | journal = ChemBioChem | volume = 13 | issue = 11 | pages = 1559–63 | date = Jul 2012 | pmid = 22730246 | doi = 10.1002/cbic.201200256 | s2cid = 13956474 }} The fluoride ions released as a result of gamma-butyrobetaine dioxygenase catalyses can be detected by using chemosensors such as protected fluorescein.{{cite journal | vauthors = Cametti M, Rissanen K | title = Recognition and sensing of fluoride anion | journal = Chemical Communications | issue = 20 | pages = 2809–29 | date = May 2009 | pmid = 19436879 | doi = 10.1039/B902069A }}

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

{{Reflist|33em}}

{{Refbegin|33em}}

• {{cite journal | vauthors = Olson AL, Rebouche CJ | title = gamma-Butyrobetaine hydroxylase activity is not rate limiting for carnitine biosynthesis in the human infant | journal = The Journal of Nutrition | volume = 117 | issue = 6 | pages = 1024–31 | date = Jun 1987 | pmid = 3110383 | doi = 10.1093/jn/117.6.1024| doi-access = free }}
• {{cite journal | vauthors = Lindstedt S, Nordin I | title = Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1) | journal = The Biochemical Journal | volume = 223 | issue = 1 | pages = 119–27 | date = Oct 1984 | pmid = 6497835 | pmc = 1144272 | doi = 10.1042/bj2230119}}
• {{cite journal | vauthors = Galland S, Le Borgne F, Bouchard F, Georges B, Clouet P, Grand-Jean F, Demarquoy J | title = Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase | journal = Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids | volume = 1441 | issue = 1 | pages = 85–92 | date = Oct 1999 | pmid = 10526231 | doi = 10.1016/s1388-1981(99)00135-3 }}
• {{cite journal | vauthors = Rigault C, Le Borgne F, Demarquoy J | title = Genomic structure, alternative maturation and tissue expression of the human BBOX1 gene | journal = Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids | volume = 1761 | issue = 12 | pages = 1469–81 | date = Dec 2006 | pmid = 17110165 | doi = 10.1016/j.bbalip.2006.09.014 }}

{{Refend}}

{{Dioxygenases}}

{{Enzymes}}

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Category:Human 2OG oxygenases

Category:EC 1.14.11

Category:Iron enzymes

Category:Ascorbate enzymes