Carnitine biosynthesis

The first enzyme of the L-carnitine biosynthetic pathway is N^ε-trimethyllysine hydroxylase, an iron and 2-oxoglutarate (2OG)-dependent oxygenase that also requires ascorbate.^[11] N^ε-trimethyllysine hydroxylase catalyses the hydroxylation reaction of N^ε-trimethyllysine to 3-hydroxy-N^ε-trimethyllysine.

The current consensus theory about the origin of N^ε-trimethyllysine in mammals is that mammals utilise lysosomal or proteasomal degradation of proteins containing N^ε-trimethyllysine residues as starting point for carnitine biosynthesis.^[12][13][14] 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.^[15] Although N^ε-trimethyllysine biosynthetic pathway involving N^ε-trimethyllysine methyltransferase has been fully characterised in fungi including Neurospora crassa, such biosynthetic pathway has never been properly characterised in mammals or humans.^[16] A third theory about the origin of N^ε-trimethyllysine in mammals does not involve biosynthesis at all, but involves direct dietary intake from vegetable foods.^{[citation needed]} High-performance liquid chromatography (HPLC) analysis has confirmed that vegetables contain a significant amount of N^ε-trimethyllysine.^[17]

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

The true identity of 3-hydroxy-N^ε-trimethyllysine aldolase is elusive and the mammalian gene encoding 3-hydroxy-N^ε-trimethyllysine aldolase has not been identified. 3-hydroxy-N^ε-trimethyllysine aldolase activity has been demonstrated in both L-threonine aldolase and serine hydroxymethyltransferase,^[18][19] although whether this is the main catalytic activity of these enzymes remains to be established.

The third enzyme of L-carnitine biosynthesis is 4-N-trimethylaminobutyraldehyde dehydrogenase.^[20] 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^ε-trimethyllysine aldolase, 4-N-trimethylaminobutyraldehyde dehydrogenase has been identified and purified from many sources including rat^[21] and Pseudomonas.^[22] 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,^[23] but the true identity of 4-N-trimethylaminobutyraldehyde dehydrogenase remains to be established.

The final step of L-carnitine biosynthesis is γ-butyrobetaine hydroxylase, a zinc binding enzyme.^{[24][25][26][27][28][29]} Like N^ε-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,^[30] rat,^[31][32][33] cow,^[34] guinea pig^[35] and human.^[36] Recombinant human γ-butyrobetaine hydroxylase has also been produced by Escherichia coli^[27] and baculoviruses^[26] systems.