Carnitine Biosynthesis Pathway Research Articles

Investigating the active site of human trimethyllysine hydroxylase.

The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate and (2S)-Nε-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242-D244-H389 residues, R391-R398 involved in 2OG binding and several residues (D231, N334 and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2S)-Nε-trimethyllysine.

Evidence That Trimethyllysine Hydroxylase Catalyzes the Formation of (2S,3S)-3-Hydroxy-Nε-trimethyllysine.

Trimethyllysine hydroxylase (TMLH) is an Fe(II) and 2-oxoglutarate (2OG) dependent oxygenase involved in the biomedically important carnitine biosynthesis pathway. A combination of synthetic and NMR studies provides direct evidence that human TMLH catalyzes the stereoselective conversion of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine.

Betaine supplementation causes increase in carnitine metabolites in the muscle and liver of mice fed a high-fat diet as studied by nontargeted LC-MS metabolomics approach

Betaine (BET) reduces diet-induced liver lipid accumulation, and may relieve obesity-related metabolic disturbances. The aim of our study was to analyze metabolite alterations after supplementation of BET, polydextrose (PDX, a soluble dietary fiber), or their combination (BET PDX) via drinking water to C57BL/6J mice fed a high-fat (HF) diet. BET supplementation increased BET levels in plasma, muscle, and liver (p < 0.05), and the nontargeted LC-MS metabolite profiling revealed an increase in several metabolites in the carnitine biosynthesis pathway after BET supplementation both in liver and muscle. These included carnitine and acetylcarnitine (1.4-fold, p < 0.05), propionylcarnitine and γ-butyrobetaine (1.5-fold, p < 0.05), and several other short-chain acylcarnitines (p < 0.05) in muscle. These changes were slightly higher in the BET PDX group. Furthermore, BET reduced the HF diet induced accumulation of triglycerides in liver (p < 0.05). The supplementations did not attenuate the HF diet induced increase in body weight gain or the increase in adipose tissue mass. Instead, the combination of BET and PDX tended to increase adiposity. Our results suggest that increased availability of BET in different tissues, especially in muscle, after BET supplementation has an impact on carnitine metabolism, and this could further explain the link between BET and lipid metabolism.

Gene cloning and biochemical characterization of 4-N-trimethylaminobutyraldehyde dehydrogenase II from Pseudomonas sp. 13CM

The gene encoding 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABaldehyde-DH) from Pseudomonas sp. 13CM, responsible for the conversion of 4-N-trimethylaminobutyraldehyde (TMABaldehyde) to γ-butyrobetaine in the carnitine biosynthesis pathway, isolated by shotgun cloning and expressed in Escherichia coli DH5α. The recombinant TMABaldehyde-DH was purified 19.5 fold to apparent homogeneity by hydrophobic and affinity chromatography and biochemically characterized. The enzyme was found to be a trimer with identical 52 kDa subunits. The isoelectric point was found to be 4.5. Optimum pH and temperature were found respectively as pH 9.5 and 40 °C. The Km values for TMABaldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were respectively, 0.31, 0.62, and 1.16 mM. The molecular and catalytic properties differed from those of TMABaldehyde-DH I, which was discovered initially in Pseudomonas sp. 13CM. The new enzyme, designated TMABaldehyde-DH II, structural gene was inserted into an expression vector pET24b (+) and over-expressed in E. coli BL21 (DE3) under the control of a T7 promoter. The recombinant TMABaldehyde-DH from Pseudomonas sp. 13CM can now be obtained in large quantity necessary for further biochemical characterization and applications.

Enzymology of the carnitine biosynthesis pathway

The water-soluble zwitterion carnitine is an essential metabolite in eukaryotes required for fatty acid oxidation as it functions as a carrier during transfer of activated acyl and acetyl groups across intracellular membranes. Most eukaryotes are able to synthesize carnitine endogenously, besides their capacity to take up carnitine from the diet or extracellular medium through plasma membrane transporters. This review discusses the current knowledge on carnitine homeostasis with special emphasis on the enzymology of the four steps of the carnitine biosynthesis pathway.

Identification and characterization of a complete carnitine biosynthesis pathway inCandida albicans

Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde dehydrogenase), and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic activities and were unable to utilize fatty acids, acetate, or ethanol as a sole carbon source, in accordance with the strict requirement for carnitine-mediated transport under these growth conditions. The second enzyme of carnitine biosynthesis, hydroxytrimethyllysine aldolase, is encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on nonfermentable carbon sources is restored only by carnitine biosynthesis intermediates after the predicted enzymatic block in the pathway, which provides independent evidence for a specific defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA family member is mainly involved in the aldolytic cleavage of hydroxytrimethyllysine in vivo.

Dietary mildronate supplementation has no effect on carnitine biosynthetic enzyme mRNA expression in rat

Abstract Mildronate (3-(2,2,2-trimethylhydrazinium)propionate) has been shown to inhibit the activity of γ -butyrobetaine hydroxylase (BBH), the last enzyme in the pathway of carnitine biosynthesis. The purpose of this study was to determine if mildronate administration to rats affects the gene expression of BBH or trimethyllysine hydroxylase (TMLH) or the activity of TMLH. Male weanling rats received control diet or control diet supplemented with 1 mg mildronate/g diet. Rats receiving mildronate had significantly depressed hepatic BBH activity relative to controls ( P γ -butyrobetaine excretion and tissue concentrations were significantly elevated in the mildronate-treated rats relative to the control animals. Trimethyllysine tissue concentrations were unaltered by mildronate administration, whereas trimethyllysine excretion was depressed relative to the controls. Thus, the effect of mildronate to inhibit hepatic BBH enzyme activity is not related to changes in the gene expression of BBH. In addition, there was no effect of mildronate upon renal TMLH activity. We conclude that mildronate affects neither BBH nor TMLH at the transcriptional level.

Effect of aromatic ring–containing drugs on carnitine biosynthesis in rats with special regard to p-aminomethylbenzoic acid

Secondary carnitine deficiencies are associated with metabolic disorders or may be the consequence of the side effects of some drugs. The mechanisms may be either a facilitated urinary excretion or an inhibited biosynthesis. Based on our earlier findings with drugs and benzoic acid analogue metabolites, in the present study, we studied the possible inhibitory effect of some benzoic acid analogue drugs. In the pathway of carnitine biosynthesis, we tested the last step, the hydroxylation of γ-butyrobetaine (Bu) to carnitine in the liver. (Liver is the only organ in rats where this step takes place.) Of the 5 tested compounds, the p-aminomethylbenzoic acid (PAMBA) was found to be inhibitory. In tracer experiments with radioactive Bu, PAMBA (a single injection of 1.2 mmol/kg) reduced the conversion of [Me- 3H]Bu to [Me- 3H]carnitine from 62.6% ± 5.11% to 46.8% ± 5.02% (means ± SEM, P < .02). This single dose also markedly reduced the conversion of loading amount of exogenous unlabeled Bu, as measured by enzymatic analysis of carnitine. The conversion of endogenous Bu was also hampered by long-term administration of PAMBA, as indicated by increased Bu and decreased carnitine levels. Furthermore, single injection of PAMBA markedly reduced the Glu level in the liver from 2.87 ± 0.17 to 1.42 ± 0.11 μmol/g ( P < .001). Trying to get closer to a mechanism by which the flux through the Bu hydroxylase was depressed, we supposed that alfa-ketoglutarate ( α-KG), an obligatory cofactor of the enzyme, was also be depressed. It was expected because α-KG is a reversible copartner of l-glutamate through the Glu-dehydrogenase reaction. We found that PAMBA reduced the α-KG level from 207 ± 17.5 to 180 ± 19.1 nmol/g (means ± SEM, P < .02). Considering the conditions of the enzyme in vitro and in vivo, this decrease may contribute to the decreased in vivo flux through the butyrobetaine hydroxylase enzyme.

L-Carnitine is Synthesized in the Human Fetal–Placental Unit: Potential Roles in Placental and Fetal Metabolism

Carnitine plays an indispensable role in fatty acid oxidation. Previous studies revealed that fetal carnitine is derived from the mother via transplacental transfer. Recent studies demonstrated the presence and importance of an active fatty acid oxidation system in the human placenta and in the human fetus. In view of these findings we decided to study carnitine metabolism in the fetal-placental unit by measuring carnitine metabolites, intermediary metabolites of carnitine biosynthesis, as well as the activity of carnitine biosynthesis enzymes in human term placenta, cord blood and selected embryonic and fetal tissues (5-20 weeks of development). Placenta contained low but detectable activity of gamma-butyrobetaine dioxygenase. This enzyme, which was considered to be expressed only in kidney, liver and brain, catalyzes the last step in the carnitine biosynthesis pathway. In addition, our results show that human fetal kidney, liver and spinal cord already have the capacity to synthesize carnitine. The ability of the placenta and fetus to synthesize carnitine suggests that in circumstances when maternal carnitine supply is limited, carnitine biosynthesis by the fetal-placental unit may supply sufficient carnitine for placental and fetal metabolism.

Contribution of a high dose of L-ascorbic acid to carnitine synthesis in guinea pigs fed high-fat diets.

Ascorbate is a cofactor of two-enzyme hydroxylation in the pathway of carnitine biosynthesis. The purpose of this study was to investigate the contribution of ascorbate to endogenous carnitine in guinea pigs fed high-fat diets. The contents of carnitine in plasma, urine and tissues of guinea pigs supplemented with L-ascorbic acid were determined and compared with those supplemented with carnitine. Albino-Hartley guinea pigs were fed vitamin C-deficient diets containing lard throughout the experiment. They were administered orally with 5 mg L-ascorbic acid/d/animal for 14 d, and then divided into three groups and administered orally with the following supplements (/d/animal) for 14 d; L (5 mg L-ascorbic acid), LASA (100 mg L-ascorbic acid), and LCAR (10 mg carnitine plus 5 mg L-ascorbic acid). As a control, a normal group was fed vitamin C-deficient diets and administered orally with 5 mg L-ascorbic acid/d/animal for 28 d. The animals fed high-fat diets (L group) had higher free-carnitine contents in the muscle and urine than the normal group. The groups of LCAR and LASA had significantly higher contents of acid-soluble carnitine (p < 0.05) in plasma than the L group. Urinary excretion of carnitine in the LASA group was decreased to the same level as that in the normal group, although no significant difference between the groups of L and LCAR was observed. Moreover, the supplement of ascorbic acid, but not of carnitine, induced a significantly lower content of triacylglycerol in the plasma of the LASA group as compared to the L group (p < 0.05). These data suggest that high doses of ascorbic acid in guinea pigs fed high-fat diets contribute to the enhancement of carnitine synthesis and improvement of the triacylglycerol content in the plasma.

The ability of guinea pigs to synthesize carnitine at a normal rate from ϵ- N-trimethyllysine or γ-butyrobetaine in vivo is not compromised by experimental vitamin C deficiency

Experimental vitamin C deficiency in guinea pigs is associated with low carnitine concentrations in blood and some tissues. Ascorbic acid is a cofactor for two enzymes in the pathway of carnitine biosynthesis. The effect of experimental vitamin C deficiency on the ability of guinea pigs to synthesize carnitine was investigated in animals fed a vitamin C-deficient diet for 28 days. On days 19 to 28, supplements (0.5 mmol · kg body weight −1 · d −) of the carnitine precursors ϵ- N-trimethyllysine or γ-butyrobetaine were administered orally. Ascorbate-supplemented, ascorbate-deficient, and pair-fed (to ascorbate-deficient) animals showed an increase in the rate of carnitine biosynthesis (as estimated from measured rates of carnitine excretion) of 32 to 40 μmol · kg body weight −1 · d −1 following supplementation with ϵ- N-trimethyllysine. Likewise, animals in each experimental group showed an increase in the rate of carnitine biosynthesis of 41 to 50 μmol · kg body weight −1 · d −1 after supplementation with γ-butyrobetaine. These results indicate that scorbutic guinea pigs are able to synthesize carnitine at a normal or above-normal rate. For guinea pigs not given a carnitine precursor supplement, rates of free and total carnitine excretion for ascorbate-deficient (but not pair-fed) animals were threefold higher than for ascorbate-supplemented animals during days 19 to 28 of the feeding regimen. Thus, carnitine depletion in vitamin C deficiency likely is due to excessive urinary excretion of carnitine and not to a decreased rate of carnitine biosynthesis.

Ascorbic acid and carnitine biosynthesis

It has been suggested that early features of scurvy (fatigue and weakness) may be attributed to carnitine deficiency. Ascorbate is a cofactor for two alpha-ketoglutarate-requiring dioxygenase reactions (epsilon-N-trimethyllysine hydroxylase and gamma-butyrobetaine hydroxylase) in the pathway of carnitine biosynthesis. Carnitine concentrations are variably low in some tissues of scorbutic guinea pigs. Ascorbic acid deficiency in guinea pigs resulted in decreased activity of hepatic gamma-butyrobetaine hydroxylase and renal but not hepatic epsilon-N-trimethyllsine hydroxylase when exogenous substrates were provided. It remains unclear whether vitamin C deficiency has a significant impact on the overall rate of carnitine synthesis from endogenous substrates. Nevertheless, results of studies of enzyme preparations and perfused liver in vitro and of scorbutic guinea pigs in vivo provide compelling evidence for participation of ascorbic acid in carnitine biosynthesis.

Hepatic synthesis of carnitine from protein-bound trimethyl-lysine. Lysosomal digestion of methyl-lysine-labelled asialo-fetuin.

The biosynthesis of carnitine in the rat was studied by following the metabolism of two radioactive derivatives of asialo-fetuin. The first contained 14C-labelled methyl groups covalently bound to the 6-N-amino fraction of its lysine residues as 6-N-monomethyl- and dimethyl-lysine. By treating this protein with iodomethane, a second derivative was produced in which the radioactivity was preferentially incorporated as 6-N-[Me-14C]-trimethyl-lysine. These desialylated glycoproteins, like other asialo-proteins, were immediately cleared from the blood by rat liver. Within hepatocyte lysosomes, the 14C-labelled proteins were rapidly hydrolysed, producing free amino acids containing the various 6-N-[Me-14C]methylated lysine residues. The radioactive amino acids crossed the lysosomal membrane and were further metabolized in the cytosol. Carnitine was the major radioactive metabolite detected in extracts of the rat carcass and liver after intravenous injection of 6-N-[Me-14C]trimethyl-lysine-labelled asialo-fetuin. Within 3h, at least 34.6% of the trimethyl-lysine in the administered protein was converted into carnitine. Similarly, an isolated perfused rat liver converted 30% of the added peptide-bound trimethyl-lysine into carnitine within 90 min. On the other hand, in numerous attempts we failed to detect radioactive carnitine in both rat liver and carcass between 20 min and 22 h after injection of 6-N-[Me-14C]-monomethyl- and -dimethyl-lysine-labelled asialo-fetuin. These data provide evidence for a pathway of carnitine biosynthesis that involves trimethyl-lysine as a peptide-bound precursor as proposed by R.A. Cox & C.L. Hoppel [(1973) Biochem. J. 136, 1083-1090] and V. Tanphaichitr & H.P. Broquist [(1973) J. Biol. Chem. 248, 2176-2181]. The findings also show that rat liver can synthesize carnitine without the aid of other tissues, but cannot convert free partially methylated lysines into trimethyl-lysine.

Biosynthesis of carnitine and 4-N-trimethylaminobutyrate from 6-N-trimethyl-lysine.

The conversion of 6-N-[Me-(14)C]trimethyl-lysine into carnitine and 4-N-trimethylaminobutyrate (butyrobetaine) was demonstrated in rats kept on a lysine-deficient diet. After the rats were given [(14)C]trimethyl-lysine for 4 days, a total of 17% of the injected label was recovered as carnitine from carcass and urine extracts. Another 8% of the trimethyl-lysine label was converted into 4-N-trimethylaminobutyrate, most of which was recovered from the urine. The conversion of trimethyl-lysine into the above two metabolites supports the pathway of carnitine biosynthesis as lysine+methionine --> 6-N-trimethyl-lysine --> 4-N-trimethylaminobutyrate --> carnitine. In addition, three other metabolites representing 2% of the injected dose were recovered. Only an insignificant portion of the label was recovered as free trimethyl-lysine from the carcass, whereas 22% of the injected label was recovered in the urine. A relatively low specific radioactivity in carnitine was found when 5-N-[Me-(14)C]trimethylaminopentanoate and 6-N-[Me-(14)C]trimethylaminohexanoate were administered to rats in amounts similar to the [(14)C]trimethyl-lysine, suggesting that they were not free intermediates.