Carnitine biosynthesis in mammals
Fre! de! ric M. VAZ1 and Ronald J. A. WANDERS
Laboratory for Genetic Metabolic Diseases, Departments of Clinical Chemistry and Paediatrics, Emma Children's Hospital, Academic Medical Centre, University of
Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands
Carnitine is indispensable for energy metabolism, since it enablesactivated fatty acids to enter the mitochondria, where they are broken down via b-oxidation. Carnitine is probably present in all animal species, and in numerous micro-organisms and plants. In mammals, carnitine homoeostasis is maintained by endogenous synthesis, absorption from dietary sources and efficient tubular reabsorption by the kidney. This review aims to cover the current
knowledge of the enzymological, molecular, metabolic and regulatory aspects of mammalian carnitine biosynthesis, with an
emphasis on the human and rat.
INTRODUCTION
Carnitine (l-3-hydroxy-4-N,N,N-trimethylaminobutyrate) is an essential metabolite, which has a number of indispensable roles in intermediary metabolism. First, carnitine has an important role in the transport of activated long-chain fatty acids from the cytosol to the mitochondrial matrix, where b-oxidation takes place (Figure 1) [1,2]. Secondly, carnitine is involved in the transfer of the products of peroxisomal b-oxidation, including acetyl-CoA, to the mitochondria for oxidation to CO# and H#O in the Krebs cycle [3,4]. Other functions of carnitine include modulation of the acyl-CoA}CoA ratio [1,5], storage of energy as acetylcarnitine [6,5] and the modulation of toxic effects of poorly metabolized acyl groups by excreting them as carnitine esters [7,8]. Carnitine is present in most, if not all, animal species, and in several micro-organisms and plants [9±12]. Animal tissues contain relatively high amounts of carnitine, varying between 0.2 and 6 lmol[g−", with the highest concentrations in heart and skeletal muscle [6]. Although animals obtain carnitine primarily from the diet, most mammals are capable of synthesizing carnitine endogenously.
Carnitine is synthesized ultimately from the amino acids lysine and methionine. Lysine provides the carbon backbone of carnitine [13,14] and the 4-N-methyl groups originate from methionine [15]. In mammals, certain proteins contain N'-
trimethyl-lysine (TML) residues [16]. N-methylation of these lysine residues occurs as a post-translational event in proteins such as calmodulin, myosin, actin, cytochrome c and histones [17,18]. This reaction is catalysed by speci®c methyltransferases, which use S-adenosylmethionine as a methyl donor [16]. Lysosomal hydrolysis of these proteins results in the release of TML, which is the ®rst metabolite of carnitine biosynthesis [19,20]. TML is ®rst hydroxylated on the 3-position by TML dioxygenase (TMLD; EC 1.14.11.8) to yield 3-hydroxy-TML (HTML). Aldolytic cleavage of HTML yields 4-trimethylaminobutyraldehyde (TMABA) and glycine, a reaction catalysed by HTML aldolase (HTMLA; EC 4.1.2.`X'). Dehydrogenation of TMABA by TMABA dehydrogenase (TMABA-DH; EC 1.2.1.47) results in the formation of 4-Ntrimethylaminobutyrate (butyrobetaine). In the last step, butyrobetaine is hydroxylated on the 3-position by c-butyrobetaine dioxygenase (BBD; EC 1.14.11.1) to yield carnitine. The chemical structure of the intermediates and the enzymes of carnitine biosynthesis are shown in Figures 2(A) and 2(B) respectively.
Because an up-to-date review on carnitine biosynthesis does not exist, while in the past few years the knowledge concerning this pathway has expanded considerably, a review on this topic is required and warranted. The present review aims to describe the current knowledge on carnitine biosynthesis at the enzymological, molecular and metabolic level. First, the individual enzymes of the carnitine-biosynthesis pathway will be discussed, including the recent developments concerning the identi®cationof the genes involved. Secondly, we will discuss the various metabolites of the carnitine-biosynthesis pathway, with an emphasis on their occurrence in biological ¯uids and on the means employed to determine their concentration. Thirdly, an overview of carnitine biosynthesis will be given for the human and rat.
Finally, the transport of carnitine and its precursors will be discussed.
Figure 1 Function of carnitine in the transport of mitochondrial long-chain fatty acid oxidation and regulation of the intramitochondrial acyl-CoA/CoA ratio
Cytosolic long-chain fatty acids, which are present as CoA esters, are trans-esteri®ed to L-carnitine in a reaction catalysed by carnitine palmitoyltransferase I (CPT I) at the mitochondrial outer
membrane. In this reaction, the acyl moiety of the long-chain fatty acids is transferred from CoA to the hydroxyl group of carnitine. The resulting long-chain acylcarnitine esters are transported
over the inner mitochondrial membrane via a speci®c carrier, carnitine-acylcarnitine translocase (CACT). At the matrix side of the mitochondrial membrane, the long-chain fatty acids are transesteri
®ed to intramitochondrial CoA, a reaction catalysed by carnitine palmitoyltransferase II (CPT II). The released carnitine can then leave the mitochondrion via CACT for another round of transport
[1]. In the mitochondrial matrix, the enzyme carnitine acetyltransferase (CAT) is able to reconvert short- and medium-chain acyl-CoAs into acylcarnitines using intramitochondrial carnitine. These
acylcarnitines can then leave the mitochondria via CACT. Through this mechanism of reversible acylation, carnitine is able to modulate the intracellular concentrations of free CoA and acyl-CoA.
Abbreviations used: ALDH9, aldehyde dehydrogenase 9; BBD, c-butyrobetaine dioxygenase; CDSP, primary systemic carnitine de®ciency; (H)TML,
(3-hydroxy-)N6-trimethyl-lysine ; HTMLA, HTML aldolase; JVS, juvenile steatosis ; OCTN2, organic cation transporter 2; PPARa, peroxisome-proliferatoractivated
receptor a; SHMT, serine hydroxymethyltransferase; TMABA, 4-N-trimethylaminobutyraldehyde; TMABA-DH, TMABA dehydrogenase; TMLD,
TML dioxygenase.
1 To whom correspondence should be addressed (e-mail f.m.vaz!amc.uva.nl).
Fre! de! ric M. VAZ1 and Ronald J. A. WANDERS
Laboratory for Genetic Metabolic Diseases, Departments of Clinical Chemistry and Paediatrics, Emma Children's Hospital, Academic Medical Centre, University of
Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands
Carnitine is indispensable for energy metabolism, since it enablesactivated fatty acids to enter the mitochondria, where they are broken down via b-oxidation. Carnitine is probably present in all animal species, and in numerous micro-organisms and plants. In mammals, carnitine homoeostasis is maintained by endogenous synthesis, absorption from dietary sources and efficient tubular reabsorption by the kidney. This review aims to cover the current
knowledge of the enzymological, molecular, metabolic and regulatory aspects of mammalian carnitine biosynthesis, with an
emphasis on the human and rat.
INTRODUCTION
Carnitine (l-3-hydroxy-4-N,N,N-trimethylaminobutyrate) is an essential metabolite, which has a number of indispensable roles in intermediary metabolism. First, carnitine has an important role in the transport of activated long-chain fatty acids from the cytosol to the mitochondrial matrix, where b-oxidation takes place (Figure 1) [1,2]. Secondly, carnitine is involved in the transfer of the products of peroxisomal b-oxidation, including acetyl-CoA, to the mitochondria for oxidation to CO# and H#O in the Krebs cycle [3,4]. Other functions of carnitine include modulation of the acyl-CoA}CoA ratio [1,5], storage of energy as acetylcarnitine [6,5] and the modulation of toxic effects of poorly metabolized acyl groups by excreting them as carnitine esters [7,8]. Carnitine is present in most, if not all, animal species, and in several micro-organisms and plants [9±12]. Animal tissues contain relatively high amounts of carnitine, varying between 0.2 and 6 lmol[g−", with the highest concentrations in heart and skeletal muscle [6]. Although animals obtain carnitine primarily from the diet, most mammals are capable of synthesizing carnitine endogenously.
Carnitine is synthesized ultimately from the amino acids lysine and methionine. Lysine provides the carbon backbone of carnitine [13,14] and the 4-N-methyl groups originate from methionine [15]. In mammals, certain proteins contain N'-
trimethyl-lysine (TML) residues [16]. N-methylation of these lysine residues occurs as a post-translational event in proteins such as calmodulin, myosin, actin, cytochrome c and histones [17,18]. This reaction is catalysed by speci®c methyltransferases, which use S-adenosylmethionine as a methyl donor [16]. Lysosomal hydrolysis of these proteins results in the release of TML, which is the ®rst metabolite of carnitine biosynthesis [19,20]. TML is ®rst hydroxylated on the 3-position by TML dioxygenase (TMLD; EC 1.14.11.8) to yield 3-hydroxy-TML (HTML). Aldolytic cleavage of HTML yields 4-trimethylaminobutyraldehyde (TMABA) and glycine, a reaction catalysed by HTML aldolase (HTMLA; EC 4.1.2.`X'). Dehydrogenation of TMABA by TMABA dehydrogenase (TMABA-DH; EC 1.2.1.47) results in the formation of 4-Ntrimethylaminobutyrate (butyrobetaine). In the last step, butyrobetaine is hydroxylated on the 3-position by c-butyrobetaine dioxygenase (BBD; EC 1.14.11.1) to yield carnitine. The chemical structure of the intermediates and the enzymes of carnitine biosynthesis are shown in Figures 2(A) and 2(B) respectively.
Because an up-to-date review on carnitine biosynthesis does not exist, while in the past few years the knowledge concerning this pathway has expanded considerably, a review on this topic is required and warranted. The present review aims to describe the current knowledge on carnitine biosynthesis at the enzymological, molecular and metabolic level. First, the individual enzymes of the carnitine-biosynthesis pathway will be discussed, including the recent developments concerning the identi®cationof the genes involved. Secondly, we will discuss the various metabolites of the carnitine-biosynthesis pathway, with an emphasis on their occurrence in biological ¯uids and on the means employed to determine their concentration. Thirdly, an overview of carnitine biosynthesis will be given for the human and rat.
Finally, the transport of carnitine and its precursors will be discussed.
Figure 1 Function of carnitine in the transport of mitochondrial long-chain fatty acid oxidation and regulation of the intramitochondrial acyl-CoA/CoA ratio
Cytosolic long-chain fatty acids, which are present as CoA esters, are trans-esteri®ed to L-carnitine in a reaction catalysed by carnitine palmitoyltransferase I (CPT I) at the mitochondrial outer
membrane. In this reaction, the acyl moiety of the long-chain fatty acids is transferred from CoA to the hydroxyl group of carnitine. The resulting long-chain acylcarnitine esters are transported
over the inner mitochondrial membrane via a speci®c carrier, carnitine-acylcarnitine translocase (CACT). At the matrix side of the mitochondrial membrane, the long-chain fatty acids are transesteri
®ed to intramitochondrial CoA, a reaction catalysed by carnitine palmitoyltransferase II (CPT II). The released carnitine can then leave the mitochondrion via CACT for another round of transport
[1]. In the mitochondrial matrix, the enzyme carnitine acetyltransferase (CAT) is able to reconvert short- and medium-chain acyl-CoAs into acylcarnitines using intramitochondrial carnitine. These
acylcarnitines can then leave the mitochondria via CACT. Through this mechanism of reversible acylation, carnitine is able to modulate the intracellular concentrations of free CoA and acyl-CoA.
Abbreviations used: ALDH9, aldehyde dehydrogenase 9; BBD, c-butyrobetaine dioxygenase; CDSP, primary systemic carnitine de®ciency; (H)TML,
(3-hydroxy-)N6-trimethyl-lysine ; HTMLA, HTML aldolase; JVS, juvenile steatosis ; OCTN2, organic cation transporter 2; PPARa, peroxisome-proliferatoractivated
receptor a; SHMT, serine hydroxymethyltransferase; TMABA, 4-N-trimethylaminobutyraldehyde; TMABA-DH, TMABA dehydrogenase; TMLD,
TML dioxygenase.
1 To whom correspondence should be addressed (e-mail f.m.vaz!amc.uva.nl).
ENZYMES OF CARNITINE BIOSYNTHESIS
Several of the carnitine-biosynthesis enzymes have been isolated and characterized, although identi®cation of the encoding genes has been realized only relatively recently [21±24]. The enzymes involved in carnitine biosynthesis, their cofactors and subcellular localization are depicted in Figure 2(B), and discussed below.
TMLD
Hulse and co-workers [25] were the ®rst to demonstrate that rat liver mitochondria are capable of hydroxylating TML to produce HTML. The enzyme responsible for this conversion was shown to be a non-haem ferrous-iron dioxygenase, which requires 2-oxoglutarate, Fe#+ and molecular oxygen as cofactors [25±28].
In this class of enzymes, the hydroxylation of the substrate is linked to the oxidative decarboxylation of 2-oxoglutarate to succinate and CO#. Molecular oxygen reacts at the active site of the enzyme to form an oxo-ferryl intermediate (Fe%+?O), and this iron-bound oxygen atom is used to hydroxylate the substrate.
The other oxygen atom is incorporated into 2-oxoglutarate, resulting in the formation of succinate and the release of CO# [29]. TMLD requires the presence of ascorbate (vitamin C) for enzymic activity, presumably to maintain the iron in the ferrous state. Reducing agents other than ascorbate are also effective (dithiothreitol, 3-mercaptoethanol), but ascorbate works best in each of the reactions [25,30].
In most experiments, TMLD activity is measured by using radiolabelled TML and counting the radioactivity of the product HTML after its isolation from the incubation medium by ionexchange chromatography [25,28,30±33]. An alternative assay was reported by Davis [24], who used unlabelled TML and detected the product (HTML), after ion-exchange chromatography, by reversed-phase HPLC using pre-column derivative formation with o-phthalaldehyde. A new method was developed recently to measure the concentration of the carnitine-biosynthesis metabolites in urine using tandem MS, and this was used to measure TMLD activity in tissue homogenates. In both humans and rats, TMLD activity is present in liver, skeletal muscle, heart and brain, but the highest activity is found in the kidney [28,31]. TMLD was puri®ed previously from bovine kidney by Henderson and co-workers [30,33], who reported that the pure enzyme was very unstable, losing all activity overnight.TMLDhas been puri®ed recently from rat kidney, and it was found that the presence of 2 mM ascorbate, 5 mM dithiothreitol and 100 g[l−" glycerol was essential for preserving the enzymic activity during the later puri®cation steps and subsequent storage at ®80 °C [24]. TMLD was characterized kinetically, and gel-®ltration and blue native PAGE analysis showed that the native enzyme is a homodimer with a mass of approx. 87 kDa [24]. The sequence of two internal peptides of the puri®ed enzyme was determined by quadruple time-of-¯ight MS. This sequence information, in combination with the data available in the expressed sequence tag database, led to the identi®cation of a rat cDNA of 1218 bp encoding a polypeptide of 405 amino acids with a calculated molecular mass of 47.5 kDa. Using the rat sequence, the authors also identi®ed the homologous cDNAs from human and mouse. Heterologous expression of both the rat and human cDNAs in COS cells con®rmed that they encode TMLD [24]. The human TMLD gene is localized at Xq28.
Subcellular localization experiments indicated that the enzyme is associated predominantly with mitochondria [25,27] in contrast with the other three carnitine-biosynthetic enzymes, which are cytosolic. Recently, the mitochondrial localization of TMLD was con®rmed by experiments using Nycodenz density-gradient analysis to resolve the different subcellular organelles [24]. The fact that TMLD is localized in mitochondria is remarkable, since the other three enzymes of the carnitine biosynthetic pathway are localized in the cytosol (Figure 2B). The submitochondrial localization of TMLD will have implications for the substrate- ¯ow and regulation of carnitine biosynthesis. Indeed, if TMLD is localized in the mitochondrial matrix, the existence of a transport system to shuttle its substrate (TML) and product (HTML) over the inner mitochondrial membrane would be required. In contrast, if TMLD is present in either the inner membrane space or the outer mitochondrial membrane, no transport system would be needed since the outer mitochondrial membrane is permeable for small molecules. This question needs to be resolved in the future.
teljes PDF letöltése:
http://www.biochemj.org/bj/361/0417/3610417.pdf
Several of the carnitine-biosynthesis enzymes have been isolated and characterized, although identi®cation of the encoding genes has been realized only relatively recently [21±24]. The enzymes involved in carnitine biosynthesis, their cofactors and subcellular localization are depicted in Figure 2(B), and discussed below.
TMLD
Hulse and co-workers [25] were the ®rst to demonstrate that rat liver mitochondria are capable of hydroxylating TML to produce HTML. The enzyme responsible for this conversion was shown to be a non-haem ferrous-iron dioxygenase, which requires 2-oxoglutarate, Fe#+ and molecular oxygen as cofactors [25±28].
In this class of enzymes, the hydroxylation of the substrate is linked to the oxidative decarboxylation of 2-oxoglutarate to succinate and CO#. Molecular oxygen reacts at the active site of the enzyme to form an oxo-ferryl intermediate (Fe%+?O), and this iron-bound oxygen atom is used to hydroxylate the substrate.
The other oxygen atom is incorporated into 2-oxoglutarate, resulting in the formation of succinate and the release of CO# [29]. TMLD requires the presence of ascorbate (vitamin C) for enzymic activity, presumably to maintain the iron in the ferrous state. Reducing agents other than ascorbate are also effective (dithiothreitol, 3-mercaptoethanol), but ascorbate works best in each of the reactions [25,30].
In most experiments, TMLD activity is measured by using radiolabelled TML and counting the radioactivity of the product HTML after its isolation from the incubation medium by ionexchange chromatography [25,28,30±33]. An alternative assay was reported by Davis [24], who used unlabelled TML and detected the product (HTML), after ion-exchange chromatography, by reversed-phase HPLC using pre-column derivative formation with o-phthalaldehyde. A new method was developed recently to measure the concentration of the carnitine-biosynthesis metabolites in urine using tandem MS, and this was used to measure TMLD activity in tissue homogenates. In both humans and rats, TMLD activity is present in liver, skeletal muscle, heart and brain, but the highest activity is found in the kidney [28,31]. TMLD was puri®ed previously from bovine kidney by Henderson and co-workers [30,33], who reported that the pure enzyme was very unstable, losing all activity overnight.TMLDhas been puri®ed recently from rat kidney, and it was found that the presence of 2 mM ascorbate, 5 mM dithiothreitol and 100 g[l−" glycerol was essential for preserving the enzymic activity during the later puri®cation steps and subsequent storage at ®80 °C [24]. TMLD was characterized kinetically, and gel-®ltration and blue native PAGE analysis showed that the native enzyme is a homodimer with a mass of approx. 87 kDa [24]. The sequence of two internal peptides of the puri®ed enzyme was determined by quadruple time-of-¯ight MS. This sequence information, in combination with the data available in the expressed sequence tag database, led to the identi®cation of a rat cDNA of 1218 bp encoding a polypeptide of 405 amino acids with a calculated molecular mass of 47.5 kDa. Using the rat sequence, the authors also identi®ed the homologous cDNAs from human and mouse. Heterologous expression of both the rat and human cDNAs in COS cells con®rmed that they encode TMLD [24]. The human TMLD gene is localized at Xq28.
Subcellular localization experiments indicated that the enzyme is associated predominantly with mitochondria [25,27] in contrast with the other three carnitine-biosynthetic enzymes, which are cytosolic. Recently, the mitochondrial localization of TMLD was con®rmed by experiments using Nycodenz density-gradient analysis to resolve the different subcellular organelles [24]. The fact that TMLD is localized in mitochondria is remarkable, since the other three enzymes of the carnitine biosynthetic pathway are localized in the cytosol (Figure 2B). The submitochondrial localization of TMLD will have implications for the substrate- ¯ow and regulation of carnitine biosynthesis. Indeed, if TMLD is localized in the mitochondrial matrix, the existence of a transport system to shuttle its substrate (TML) and product (HTML) over the inner mitochondrial membrane would be required. In contrast, if TMLD is present in either the inner membrane space or the outer mitochondrial membrane, no transport system would be needed since the outer mitochondrial membrane is permeable for small molecules. This question needs to be resolved in the future.
teljes PDF letöltése:
http://www.biochemj.org/bj/361/0417/3610417.pdf
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