Misplaced Pages

Carnitine biosynthesis is a method for the endogenous production of L-carnitine, a molecule that is essential for energy metabolism. In humans and many other animals, L-carnitine is obtained from both diet and by biosynthesis. The carnitine biosynthesis pathway is highly conserved among many eukaryotes and some prokaryotes.

L-Carnitine is biosynthesized from N-trimethyllysine. At least four enzymes are involved in the overall biosynthetic pathway. They are N-trimethyllysine hydroxylase, 3-hydroxy-N-trimethyllysine aldolase, 4-N-trimethylaminobutyraldehyde dehydrogenase and γ-butyrobetaine hydroxylase.

N-Trimethyllysine hydroxylase

The first enzyme of the L-carnitine biosynthetic pathway is N-trimethyllysine hydroxylase, an iron and 2-oxoglutarate (2OG)-dependent oxygenase that also requires ascorbate. N-trimethyllysine hydroxylase catalyses the hydroxylation reaction of N-trimethyllysine to 3-hydroxy-N-trimethyllysine.

The current consensus theory about the origin of N-trimethyllysine in mammals is that mammals utilise lysosomal or proteasomal degradation of proteins containing N-trimethyllysine residues as starting point for carnitine biosynthesis. An alternative theory involving endogenous non-peptidyl biosynthesis was also proposed, based on evidence gathered from a study involving feeding normal and undernourished human subjects with the amino acid lysine. Although N-trimethyllysine biosynthetic pathway involving N-trimethyllysine methyltransferase has been fully characterised in fungi including Neurospora crassa, such biosynthetic pathway has never been properly characterised in mammals or humans. A third theory about the origin of N-trimethyllysine in mammals does not involve biosynthesis at all, but involves direct dietary intake from vegetable foods. High-performance liquid chromatography (HPLC) analysis has confirmed that vegetables contain a significant amount of N-trimethyllysine.

3-Hydroxy-N-trimethyllysine aldolase

The second step of L-carnitine biosynthesis requires the 3-hydroxy-N-trimethyllysine aldolase enzyme. 3-hydroxy-N-trimethyllysine aldolase is a pyridoxal phosphate dependent aldolase, and it catalyses the cleavage of 3-hydroxy-N-trimethyllysine into 4-N-trimethylaminobutyraldehyde and glycine.

The true identity of 3-hydroxy-N-trimethyllysine aldolase is elusive and the mammalian gene encoding 3-hydroxy-N-trimethyllysine aldolase has not been identified. 3-hydroxy-N-trimethyllysine aldolase activity has been demonstrated in both L-threonine aldolase and serine hydroxymethyltransferase, although whether this is the main catalytic activity of these enzymes remains to be established.

4-N-Trimethylaminobutyraldehyde dehydrogenase

The third enzyme of L-carnitine biosynthesis is 4-N-trimethylaminobutyraldehyde dehydrogenase. 4-N-trimethylaminobutyraldehyde dehydrogenase is a NAD dependent enzyme. 4-N-trimethylaminobutyraldehyde dehydrogenase catalyses the dehydrogenation of 4-N-trimethylaminobutyraldehyde into gamma-butyrobetaine.

Unlike 3-hydroxy-N-trimethyllysine aldolase, 4-N-trimethylaminobutyraldehyde dehydrogenase has been identified and purified from many sources including rat and Pseudomonas. However, the human 4-N-trimethylaminobutyraldehyde dehydrogenase has so far not been identified. There is considerable sequence similarity between rat 4-N-trimethylaminobutyraldehyde dehydrogenase and human aldehyde dehydrogenase 9, but the true identity of 4-N-trimethylaminobutyraldehyde dehydrogenase remains to be established.

γ-Butyrobetaine hydroxylase

The final step of L-carnitine biosynthesis is γ-butyrobetaine hydroxylase, a zinc binding enzyme. Like N-trimethyllysine hydroxylase, γ-butyrobetaine hydroxylase is a 2-oxoglutarate and iron(II)-dependent oxygenase. γ-Butyrobetaine hydroxylase catalyses the stereospecific hydroxylation of γ-butyrobetaine to L-carnitine.

γ-Butyrobetaine hydroxylase is the most studied enzyme among the four enzymes in the biosynthetic pathway. It has been purified from many sources, such as Pseudomonas, rat, cow, guinea pig and human. Recombinant human γ-butyrobetaine hydroxylase has also been produced by Escherichia coli and baculoviruses systems.

References

1. Activation and Transportation of Fatty Acids for Metabolism via Carnitine Shuttle Pathway (with Animation)
2. Fraenkel, G.; Friedman, S. Carnitine. Vitam. Horm. 1957, 15, 73–118.
3. Bremer, J. Carnitine – metabolism and functions. Physiol. Rev. 1983, 63, 1420–1480.
4. Steiber, A.; Kerner, J.; Hoppel, C. L. Carnitine: a nutritional, biosynthetic, and functional perspective. Mol. Aspects Med. 2004, 25, 455–473.
5. Rebouche, C. J. Carnitine function and requirements during the life cycle. FASEB J. 1992, 6, 3379–3386.
6. Lennon, D. L.; Shrago, E. R.; Madden, M.; Nagle, F. J.; Hanson, P. Dietary carnitine intake related to skeletal muscle and plasma carnitine concentrations in adult men and women. Am. J. Clin. Nutr. 1986, 43, 234–238.
7. Lindstedt, G.; Lindstedt, S.; Midtvedt, T.; Tofft, M. The formation and degradation of carnitine in Pseudomonas. Biochemistry 1967, 6, 1262–1270.
8. Vaz, F. M.; Wanders, R. J. A. Carnitine biosynthesis in mammals. Biochem. J. 2002, 361, 417–429.
9. Strijbis, K.; Vaz, F, M.; Distel, B. Enzymology of the carnitine biosynthesis pathway. IUBMB Life 2010, 62, 357–362.
10. Hulse, J. D.; Ellis, S. R.; Henderson, L. M. Carnitine biosynthesis. β-Hydroxylation of trimethyllysine by an α-ketoglutarate-dependent mitochondrial dioxygenase. J. Biol. Chem. 1978, 253, 1654–1659.
11. Vaz, F. M.; Ofman, R.; Westinga, K.; Back, J. W.; Wanders, R. J. A. Molecular and biochemical characterization of rat ε-N-trimethyllysine hydroxylase, the first enzyme of carnitine biosynthesis. J. Biol. Chem. 2001, 276, 33512–33517.
12. Bremer, J. Biosynthesis of carnitine in vivo. Biochim. Biophys. Acta 1961, 48, 622–624.
13. Wolf, G.; Berger, C. R. A. Studies on the biosynthesis and turnover of carnitine. Arch. Biochem. Biophys. 1961, 92, 360–365.
14. Paik, W. K.; Nochumson, S.; Kim, S. Carnitine biosynthesis via protein methylation. Trends Biochem. Sci. 1977, 2, 159–161.
15. Khan-Siddiqui, L.; Bamji, M. S. Lysine-carnitine conversion in normal and undernourished adult man – suggestion of a nonpeptidyl pathway. Am. J. Clin. Nutr. 1983, 37, 93–98.
16. Rebouche, C. J.; Broquist, H. P. Carnitine biosynthesis in Neurospora crassa: enzymatic conversion of lysine to ε-N-trimethyllysine. J. Bacteriol. 1976, 126, 1207–1214.
17. Servillo, L.; Giovane, A.; Cautela, D.; Castaldo, D.; Balestrieri, M. L. Where Does N-Trimethyllysine for the Carnitine Biosynthesis in Mammals Come from? PLoS ONE 2014, 9, e84589.
18. McNeil, J. B.; Flynn, J.; Tsao, N.; Monschau, N.; Stahmann, K. P.; Haynes, R. H.; McIntosh, E. M.; Pearlman, R. E. Glycine metabolism in Candida albicans: characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes. Yeast 2000, 16, 167–175.
19. Schirch, L.; Peterson, D. Purification and properties of mitochondrial serine hydroxymethyltransferas. J. Biol. Chem. 1980, 255, 7801–7806.
20. Hulse, J. D.; Henderson, L. M. Carnitine biosynthesis. Purification of 4-N’-trimethylaminobutyraldehyde dehydrogenase from beef liver. J. Biol. Chem. 1980, 255, 1146–1151.
21. Vaz, F. M.; Fouchier, S. W.; Ofman, R.; Sommer, M.; Wanders, R. J. A. Molecular and biochemical characterization of rat γ-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis. J. Biol. Chem. 2000, 275, 7390–7394.
22. Hassan, M.; Okada, M.; Ichiyanagi, T.; Mori, N. 4-N-Trimethylaminobutyraldehyde dehydrogenase: purification and characterization of an enzyme from Pseudomonas sp. 13CM. Biosci. Biotechnol. Biochem. 2008, 72, 155–162.
23. Lin, S. W.; Chen, J. C.; Hsu, L. C.; Hsieh, C. L.; Yoshida, A. Human γ-aminobutyraldehyde dehydrogenase (ALDH9): cDNA sequence, genomic organization, polymorphism, chromosomal localization, and tissue expression. Genomics 1996, 34, 376–380
24. Vaz, F. M.; van Gool, S.; Ofman, R.; Ijlst, L.; Wanders, R. J. Carnitine Biosynthesis: Identification of the cDNA Encoding Human γ-Butyrobetaine Hydroxylase. Biochem. Biophys. Res. Commun. 1998, 250, 506–510.
25. Rigault, C.; Le Borgne, F.; Demarquoy, J. Genomic structure, alternative maturation and tissue expression of the human BBOX1 gene. Biochim. Biophys. Acta 2006, 1761, 1469–1481.
26. ^ Leung, I. K. H.; Krojer, T. J.; Kochan, G. T.; Henry, L.; von Delft, F.; Claridge, T. D. W.; Oppermann, U.; McDonough, M. A.; Schofield, C. J. Structural and mechanistic studies on γ-butyrobetaine hydroxylase. Chem. Biol. 2010, 17, 1316–1324.
27. ^ Tars, K.; Rumnieks, J.; Zeltins, A.; Kazaks, A.; Kotelovica, S.; Leonciks, A.; Saripo, J.; Viksna, A.; Kuka, J.; Liepinsh, E.; Dambrova, M. Crystal structure of human gamma-butyrobetaine hydroxylase. Biochem. Biophys. Res. Commun. 2010, 298, 634–639.
28. Lindstedt, G.; Lindstedt, S.; Olander, B.; Tofft, M. α-ketoglutarate and hydroxylation of γ-butyrobetaine. Biochim. Biophys. Acta 1968, 158, 503–505.
29. Lindstedt, G.; Lindstedt, S. Cofactor requirements of γ-butyrobetaine hydroxylase from rat liver. J. Biol. Chem. 1970, 245, 4178–4186.
30. Lindstedt, G.; Lindstedt, S.; Tofft, S. γ-Butyrobetaine hydroxylase from Pseudomonas sp AK 1. Biochemistry 1970, 9, 4336–4342
31. Lindstedt, G. Hydroxylation of γ-butyrobetaine to carnitine in rat liver. Biochemistry 1967, 6, 1271–1282.
32. Paul, H. S.; Sekas, G.; Adibi, S. A. Carnitine biosynthesis in hepatic peroxisomes. Demonstration of γ-butyrobetaine hydroxylase activity. Eur. J. Chem. 1992, 203, 599–605.
33. Galland, S.; Leborgne, F.; Guyonnet, D.; Clouet, P.; Demarquoy, J. Purification and characterization of the rat liver γ-butyrobetaine hydroxylase. Mol. Cell. Biochem. 1998, 178, 163–168.
34. Kondo, A.; Blanchard, J. S.; Englard, S. Purification and properties of calf liver γ-butyrobetaine hydroxylase. Arch. Biochem. Biophys. 1981, 212, 338–346.
35. Dunn. W. A.; Rettura, G.; Seifter, E.; Englard, S. Carnitine biosynthesis from γ-butyrobetaine and from exogenous protein-bound 6-N-trimethyl-L-lysine by the perfused guinea pig liver. J. Biol. Chem. 1984, 259, 10764–10770.
36. Lindstedt, G.; Lindstedt, S.; Nordin, I. γ-Butyrobetaine hydroxylase in human kidney. Scand. J. Clin. Lab. Invest. 1982, 42, 477–485.

• Biosynthesis