Gamma-butyrobetaine dioxygenase (also known as BBOX, GBBH or γ-butyrobetaine hydroxylase) is an
enzyme that in humans is encoded by the BBOX1gene.[5][6] Gamma-butyrobetaine dioxygenase catalyses the formation of
L-carnitine from gamma-butyrobetaine, the last step in the L-
carnitine biosynthesis pathway.[7]Carnitine is essential for the transport of activated
fatty acids across the
mitochondrial membrane during mitochondrial
beta oxidation.[6] In humans, gamma-butyrobetaine dioxygenase can be found in the kidney (high), liver (moderate), and brain (very low).[5][8]BBOX1 has recently been identified as a potential
cancergene based on a large-scale
microarray data analysis.[9]
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
2OGoxygenases, 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
2OGoxygenases 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]
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
2OGoxygenases 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]
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[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
2OGoxidation 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
expensiveinstrumentation. A potentially
high-throughputfluorescence-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]
^Nelson PJ, Pruitt RE, Henderson LL, Jenness R, Henderson LM (Jan 1981). "Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine". Biochimica et Biophysica Acta (BBA) - General Subjects. 672 (1): 123–7.
doi:
10.1016/0304-4165(81)90286-5.
PMID6783120.
^Wehbie RS, Punekar NS, Lardy HA (Mar 1988). "Rat liver gamma-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogues on hydroxylation and decarboxylation". Biochemistry. 27 (6): 2222–8.
doi:
10.1021/bi00406a062.
PMID3378057.
^PDB:
3N6W;Tars K, Rumnieks J, Zeltins A, Kazaks A, Kotelovica S, Leonciks A, Sharipo J, Viksna A, Kuka J, Liepinsh E, Dambrova M (Aug 2010). "Crystal structure of human gamma-butyrobetaine hydroxylase". Biochemical and Biophysical Research Communications. 398 (4): 634–9.
doi:
10.1016/j.bbrc.2010.06.121.
PMID20599753.
^
abHenry L, Leung IK, Claridge TD, Schofield CJ (Aug 2012). "γ-Butyrobetaine hydroxylase catalyses a Stevens type rearrangement". Bioorganic & Medicinal Chemistry Letters. 22 (15): 4975–8.
doi:
10.1016/j.bmcl.2012.06.024.
PMID22765904.
^Stevens TS, Creighton EM, Gordon AB, MacNicol M (1928). "CCCCXXIII.—Degradation of quaternary ammonium salts. Part I". J. Chem. Soc.: 3193–3197.
doi:
10.1039/JR9280003193.
^Loenarz C, Schofield CJ (Mar 2008). "Expanding chemical biology of 2-oxoglutarate oxygenases". Nature Chemical Biology. 4 (3): 152–6.
doi:
10.1038/nchembio0308-152.
PMID18277970.
^Hayashi Y, Kirimoto T, Asaka N, Nakano M, Tajima K, Miyake H, Matsuura N (May 2000). "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.
doi:
10.1016/S0014-2999(00)00098-4.
PMID10812052.
^Pupure J, Isajevs S, Skapare E, Rumaks J, Svirskis S, Svirina D, Kalvinsh I, Klusa V (Feb 2010). "Neuroprotective properties of mildronate, a mitochondria-targeted small molecule". Neuroscience Letters. 470 (2): 100–5.
doi:
10.1016/j.neulet.2009.12.055.
PMID20036318.
S2CID38603504.
^Liepinsh E, Skapare E, Svalbe B, Makrecka M, Cirule H, Dambrova M (May 2011). "Anti-diabetic effects of mildronate alone or in combination with metformin in obese Zucker rats". European Journal of Pharmacology. 658 (2–3): 277–83.
doi:
10.1016/j.ejphar.2011.02.019.
PMID21371472.
^Zvejniece L, Svalbe B, Makrecka M, Liepinsh E, Kalvinsh I, Dambrova M (Sep 2010). "Mildronate exerts acute anticonvulsant and antihypnotic effects". Behavioural Pharmacology. 21 (5–6): 548–55.
doi:
10.1097/FBP.0b013e32833d5a59.
PMID20661137.
S2CID12501700.
^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 (Oct 2013). "Efficacy and safety of mildronate for acute ischemic stroke: a randomized, double-blind, active-controlled phase II multicenter trial". Clinical Drug Investigation. 33 (10): 755–60.
doi:
10.1007/s40261-013-0121-x.
PMID23949899.
S2CID697191.
^Rose NR, McDonough MA, King ON, Kawamura A, Schofield CJ (Aug 2011). "Inhibition of 2-oxoglutarate dependent oxygenases". Chemical Society Reviews. 40 (8): 4364–97.
doi:
10.1039/c0cs00203h.
PMID21390379.
^Galland S, Le Borgne F, Guyonnet D, Clouet P, Demarquoy J (Jan 1998). "Purification and characterization of the rat liver gamma-butyrobetaine hydroxylase". Molecular and Cellular Biochemistry. 178 (1–2): 163–8.
doi:
10.1023/A:1006849713407.
PMID9546596.
S2CID23339575.
^Petter RC, Banerjee S, Englard S (1990). "Inhibition of γ-butyrobetaine hydroxylase by cyclopropyl-substituted γ-butyrobetaines". J. Org. Chem. 55 (10): 3088–3097.
doi:
10.1021/jo00297a025.
^Ziering DL, Pascal Jr RA (1990). "Mechanism-based inhibition of bacterial γ-butyrobetaine hydroxylase". J. Am. Chem. Soc. 112 (2): 834–838.
doi:
10.1021/ja00158a051.
^Lindstedt G, Lindstedt S, Tofft S (1970). "γ-Butyrobetaine Hydroxylase from Pseudomonas sp AK 1". Biochemistry. 9 (22): 4336–4342.
doi:
10.1021/bi00824a014.
PMID5472709.
^Lindstedt G, Lindstedt S, Nordin I (1977). "Purification and properties of γ-butyrobetaine hydroxylase from Pseudomonas species AK 1". Biochemistry. 16 (10): 2181–2188.
doi:
10.1021/bi00629a022.
PMID861203.
^Cederblad C, Lindstedt S (1972). "A method for the determination of carnitine in the picomole range". Clin. Chim. Acta. 37: 235–243.
doi:
10.1016/0009-8981(72)90438-X.
PMID5022087.
^Böhmer T, Rydning A, Solberg HE (1974). "Carnitine levels in human serum in health and disease". Clin. Chim. Acta. 57 (1): 55–61.
doi:
10.1016/0009-8981(74)90177-6.
PMID4279150.
^Parvin R, Pande SV (1976). "Microdetermination of (−)carnitine and carnitine acetyltransferase activity". Anal. Biochem. 79 (1–2): 190–201.
doi:
10.1016/0003-2697(77)90393-1.
PMID869176.
^Rydzik AM, Leung IK, Kochan GT, Thalhammer A, Oppermann U, Claridge TD, Schofield CJ (Jul 2012). "Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase". ChemBioChem. 13 (11): 1559–63.
doi:
10.1002/cbic.201200256.
PMID22730246.
S2CID13956474.
^Cametti M, Rissanen K (May 2009). "Recognition and sensing of fluoride anion". Chemical Communications (20): 2809–29.
doi:
10.1039/B902069A.
PMID19436879.
Galland S, Le Borgne F, Bouchard F, Georges B, Clouet P, Grand-Jean F, Demarquoy J (Oct 1999). "Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase". Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1441 (1): 85–92.
doi:
10.1016/s1388-1981(99)00135-3.
PMID10526231.
Rigault C, Le Borgne F, Demarquoy J (Dec 2006). "Genomic structure, alternative maturation and tissue expression of the human BBOX1 gene". Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1761 (12): 1469–81.
doi:
10.1016/j.bbalip.2006.09.014.
PMID17110165.