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J. Biol. Chem., Vol. 276, Issue 36, 33512-33517, September 7, 2001

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Molecular and Biochemical Characterization of Rat -N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis^*

Frédéric M. Vaz, Rob Ofman, Kasper Westinga, Jaap Willem Back^§, and Ronald J. A. Wanders^¶

From the  Laboratory for Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, P. O. Box 22700, Amsterdam 1100 DE, and the ^§ Mass Spectrometry Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1018 TV, The Netherlands

Received for publication, June 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-N-Trimethyllysine hydroxylase (EC 1.14.11.8) is the first enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of -hydroxy-N--trimethyllysine from -N-trimethyllysine, a reaction dependent on -ketoglutarate, Fe²⁺, and oxygen. We purified the enzyme from rat kidney and sequenced two internal peptides by quadrupole-time-of-flight mass spectroscopy. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA of 1218 base pairs encoding a polypeptide of 405 amino acids with a calculated molecular mass of 47.5 kDa. Using the rat sequence we also identified the homologous cDNAs from human and mouse. Heterologous expression of both the rat and human cDNAs in COS cells confirmed that they encode -N-trimethyllysine hydroxylase. Subcellular fractionation studies revealed that the rat enzyme is localized exclusively in mitochondria. Expression studies in yeast indicated that the rat enzyme is synthesized as a 47.5-kDa precursor and subsequently processed to a mature protein of 43 kDa, presumably upon import in mitochondria. The Michaelis-Menten constants of the purified rat enzyme for trimethyllysine, -ketoglutarate, and Fe²⁺ were 1.1 mM, 109 µM, and 54 µM, respectively. Both gel filtration and blue native polyacrylamide gel electrophoresis analysis showed that the native enzyme has a mass of approximately 87 kDa, indicating that in rat -N-trimethyllysine hydroxylase is a homodimer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Carnitine (3-hydroxy-4-N-trimethylaminobutyrate) is a vital compound, which plays an indispensable role in the transport of activated fatty acids across the inner mitochondrial membrane into the matrix, where -oxidation takes place (1, 2). Furthermore, carnitine is involved in the transfer of the products of peroxisomal -oxidation, including acetyl-CoA, to the mitochondria for oxidation to CO₂ and H₂O in the Krebs cycle (3, 4). Apart from the dietary intake of carnitine, most eukaryotes are able to synthesize this compound from trimethyllysine (5-7). The trimethyllysine is generated by the hydrolysis of proteins containing lysines that are trimethylated at their -amino group by a protein-dependent methyltransferase using S-adenosylmethionine as a methyl donor. In the carnitine biosynthetic pathway, trimethyllysine is first hydroxylated at the -position by -trimethyllysine hydroxylase (TMLH¹), after which the resulting -hydroxytrimethyllysine is cleaved by a specific aldolase into -trimethylaminobutyraldehyde and glycine (6, 8). Subsequently, -trimethylaminobutyraldehyde is oxidized by -trimethylaminobutyraldehyde dehydrogenase to form -butyrobetaine (9). In the last step, -butyrobetaine is hydroxylated at the -position by a second hydroxylase, -butyrobetaine hydroxylase, yielding L-carnitine (5, 7, 10). In rat and mouse, -butyrobetaine hydroxylase is localized exclusively in the liver, whereas in humans, the enzyme is present in kidney, liver, and brain. Although most tissues are capable of converting trimethyllysine into -butyrobetaine, liver and kidney are the main sites of carnitine biosynthesis in all mammals, including humans (10-15).

After the recent identification of the cDNAs coding for -trimethylaminobutyraldehyde dehydrogenase and -butyrobetaine hydroxylase (16-18), we focused our attention on the first enzyme of the carnitine biosynthesis, TMLH. Like -butyrobetaine hydroxylase, TMLH is a non-heme ferrous-iron dioxygenase that requires -ketoglutarate, Fe²⁺, and molecular oxygen as cofactors (8, 19-21). In this class of enzymes, the hydroxylation of the substrate is linked to the oxidative decarboxylation of -ketoglutarate. In both humans and rat, the highest TMLH activity is found in kidney but is also present in liver, skeletal muscle, heart, and brain (13, 20). Subcellular localization experiments using differential centrifugation indicated that the enzyme is predominantly localized in mitochondria (8, 21) in contrast to the other three carnitine biosynthetic enzymes, which are cytosolic. We purified the hydroxylase responsible for the conversion of trimethyllysine to hydroxytrimethyllysine from rat kidney and determined part of its amino acid sequence by (quadrupole-time-of-flight) mass spectrometry. Using this sequence information we identified the cDNAs encoding trimethyllysine hydroxylase from rat, human, and mouse. Finally, we expressed the rat and human cDNAs in COS cells to confirm that the identified cDNAs encode TMLH.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Trimethyllysine was purchased from Sigma Chemical Co. Q-Sepharose HP and Butyl-Sepharose 4 Fast Flow were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden), and CHT-II hydroxylapatite was from Bio-Rad (Hercules, CA). All other reagents were of analytical grade. The pMAL-C2X vector was purchased from New England BioLabs (Herts, United Kingdom), the pcDNA3 vector was from Invitrogen (San Diego, CA).

TMLH Assay-- Two methods were used to determine TMLH activity. In the first method, TMLH activity was determined radiochemically by measuring trimethyllysine-dependent release of [¹⁴C]CO₂ that is produced from the decarboxylation of -[1-¹⁴C]ketoglutarate to succinate. The assay mixture (total volume, 250 µl) contained 75 mM Tris/MES(HCl) buffer, pH 6.7, containing 90 µM -ketoglutarate, 10 µM -[1-¹⁴C]ketoglutarate, 2.5 mM sodium ascorbate, 5.0 mM calcium chloride, 0.5 mM dithiothreitol, 0.5 mM ammonium iron(II)sulfate, 2 mg/ml bovine serum albumin, and 2 mM trimethyllysine. The reaction was started by adding the enzyme sample to the reaction mixture and allowed to proceed for 30 min at 37 °C, after which it was terminated by the addition of 100 µl of perchloric acid. The released [¹⁴C]O₂ was trapped in 0.5 ml of 2 M NaOH, essentially as described by Wanders et al. (22), and the NaOH was counted for radioactivity in a liquid scintillation counter.

In the second method, the amount of hydroxytrimethyllysine that was enzymatically produced from trimethyllysine was determined by HPLC tandem MS. The reaction mixture and incubation time were the same as in the radiochemical method, except for the -ketoglutarate concentration, which was 2.5 mM instead of 0.1 mM. This method will be described in detail elsewhere. Briefly, the reaction mixture was applied on a Microcon centrifugal filter unit with a 30-kDa cut-off (Millipore, Bedford, MA) to remove most of the proteins. 100 µl of the filtrate was derivatized with methyl chloroformate at alkaline pH, followed by ethyl acetate extraction. Part of the aqueous phase was injected into an ion pair HPLC system using heptafluorobutyric acid as ion pairing agent, and the hydroxytrimethyllysine was quantified by tandem MS.

Purification of TMLH-- Kidneys were taken from male Wistar rats and homogenized by five strokes of a Teflon pestle in a Potter-Elvehjem glass homogenizer at 500 rpm in a 10 mM Tris/HCl buffer, pH 8.0, containing 100 g/liter glycerol, 100 mM KCl, and 5 mM dithiothreitol (DTT). The crude homogenate was centrifuged for 10 min at 800 × g at 4 °C to remove nuclei and whole cells. The resulting supernatant was sonicated 5 times for 30 s at 10 watts and centrifuged for 1 h at 33,000 × g at 4 °C. The supernatant was collected, and the pellet was resuspended in the same buffer, after which the sonication and centrifugation steps were repeated. The two supernatants were pooled, diluted 20-fold in a 5 mM MES buffer, pH 6.0, containing 100 g/liter glycerol and 5 mM DTT and incubated overnight at 4 °C. The resulting protein precipitate was pelleted by centrifugation for 20 min at 20,000 × g at 4 °C and dissolved in a 20 mM ethanolamine buffer, pH 9.3, containing 100 g/liter glycerol, 25 mM KCl, and 5 mM DTT. After centrifugation for 20 min at 20,000 × g at 4 °C, the supernatant was applied to a Q-Sepharose HP column (diameter = 2.6 cm; h = 10 cm; flow: 3 ml/min), which was pre-equilibrated with a 20 mM ethanolamine buffer, pH 9.3, containing 100 g/liter glycerol and 2.5 mM DTT. Bound proteins were eluted with a linear gradient from 25 to 400 mM KCl in the same buffer. Fractions containing high TMLH activity were pooled and diluted 1:4 in a 20 mM ethanolamine buffer, pH 9.3, containing 100 g/liter glycerol, 200 mM ammonium sulfate, 2 mM sodium ascorbate, 1 M KCl, and 5 mM DTT. The solution was centrifuged for 10 min at 4,000 × g at 4 °C to remove protein precipitates, and the supernatant was loaded onto a Butyl-Sepharose 4 Fast Flow column (diameter = 1.6 cm; h = 11 cm; flow: 2.5 ml/min), pre-equilibrated with the dilution-buffer. Bound proteins were eluted with a linear gradient from 1 M KCl + 200 mM ammonium sulfate to 0 M of both salts in a 20 mM ethanolamine buffer, pH 9.3, containing 100 g/liter glycerol, 2 mM sodium ascorbate, and 5 mM DTT. Fractions containing high TMLH activity were pooled and diluted 1:1 in a 20 mM ethanolamine buffer, pH 9.2, containing 100 g/liter glycerol, 2 mM sodium ascorbate, and 5 mM DTT and loaded onto an Econo-Pac CHT-II hydroxylapatite column (diameter = 2 cm; h = 5 cm; flow: 1 ml/min) equilibrated with the same buffer. Bound proteins were eluted with a linear gradient from 0 to 50 mM potassium phosphate. Fractions were tested for TMLH activity and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. SDS-PAGE and silver staining were performed as described by Laemmli (23) and Rabilloud et al. (24), respectively. Protein concentrations were determined by the method of Bradford (25), using bovine serum albumin as standard.

Characterization of the Purified TMLH-- The Michaelis-Menten constants (K_m) of purified TMLH for trimethyllysine, -ketoglutarate, and Fe²⁺ were determined using the radiometric assay described above. For the determination of the K_m of -ketoglutarate and Fe²⁺, a fixed concentration of 2 mM trimethyllysine was used. The pH optimum was determined by using 75 mM bis-tris-propane buffer instead of the Tris/MES buffer, at pH values ranging from 5.5 to 9.5 in steps of 1 pH unit.

Protein and Peptide Analysis-- For MALDI-TOF MS analysis, protein-containing gel slices were S-alkylated, digested with trypsin (Roche Molecular Biochemicals, sequencing grade), and extracted according to Shevchenko et al. (26). Extracted peptides were purified and concentrated using Zip-Tips (Millipore). Peptides were eluted from the Zip-Tips with 10 µl of 1% (v/v) formic acid, 60% acetonitrile. The peptide solution was mixed with an equal volume of 10 mg/ml -cyano-4-hydroxycinnamic acid (Sigma Chemical Co.) solution in acetonitrile/ethanol (1:1, v/v). Aliquots of 0.5 µl were spotted on the target and allowed to dry at room temperature. MALDI-TOF MS spectra were acquired on a Micromass TofSpec 2EC (Micromass, Wythenshawe, UK) equipped with a 2-GHz digitizer. The resulting peptide spectra were used to search a non-redundant protein sequence data base (Swiss-Prot/TREMBL) using the Proteinprobe program. For ESI-Q-TOF MS the peptide solution (2 µl) was introduced into a nanospray capillary, and positive mode spectra were recorded with a Q-TOF mass spectrometer (Micromass) equipped with a Z-spray source.

Cloning and Expression of TMLH in COS Cells-- The complete open reading frame (ORF) of rat TMLH was amplified by the polymerase chain reaction (PCR) from rat kidney cDNA using Pwo DNA polymerase (Roche Molecular Biochemicals) and the following primers: a BamHI-tagged forward primer 5'-aaaggatccATGAAGAGAGGAGACATAGCTCAC-3' and a NotI-tagged reverse primer 5'-ttttgcggccgcTTAGGCATGAAGACCTAGAATTC-3'. The human ORF of TMLH was amplified from human kidney cDNA using the following primers: a BamHI-tagged forward primer 5'-tataggatccATGTGGTACCACAGATTGTC-3' and an NotI-tagged reverse primer 5'-tatagcggccgcCTGTTAAGCCTGAAGCCCCAAGA-3'. The PCR products were cloned downstream of the P_CMV promoter into the BamHI and NotI sites of the mammalian expression vector pcDNA3. Both ORFs were sequenced to exclude sequence errors introduced by Pwo DNA polymerase during the PCR, after which the constructs were transfected to COS cells using the LipofectAMINE Plus reagent (Life Technologies, Rockville, MD) as described by the manufacturer. 48 h after transfection, cells were harvested by trypsinization and lysed in a 10 mM sodium phosphate buffer, pH 7.4, containing 140 mM NaCl, 200 g/liter glycerol, and 1 mM DTT by sonicating two times for 15 s at 8 watts. TMLH activity was determined by the HPLC tandem MS method described above.

TMLH Expression in Yeast-- The rat and human TMLH ORFs were amplified as described above using the same primers. For the amplification of the rat ORF starting from the second methionine, the following BamHI-tagged forward primer was used: 5'-ttttggatccATGCGCTTTGATTATGTCTGGC-3' in combination with the same reverse primer described above. The PCR products were cloned downstream of the galactose-inducible GAL1 promoter into the BamHI and NotI sites of the yeast expression vector pYES2. To assess the fidelity of the PCR process the ORFs were sequenced. The constructs were transformed to the Saccharomyces cerevisiae strain INVSC2 using the lithium acetate procedure (27). Transformed yeast cells were grown on minimal glucose medium (6.7 g/liter yeast nitrogen base, 3 g/liter glucose) to fully repress transcription of the GAL1 promoter. Cells were transferred to minimal lactate medium (6.7 g/liter yeast nitrogen base, 20 g/liter lactate) and galactose was added to a final concentration of 4 g/liter to induce protein expression. After overnight induction, spheroplasts were prepared using zymolyase (ICN Biomedicals, Costa Mesa, CA) according to Franzusoff et al. (28) and lysed in a 10 mM sodium phosphate buffer, pH 7.4, containing 140 mM NaCl, 200 g/liter glycerol, and 1 mM DTT.

TMLH Antibody Generation-- The complete ORF of TMLH was amplified by PCR from human kidney cDNA using Pwo DNA polymerase and the following primers: a BamHI-tagged forward primer 5'-tataggatccATGTGGTACCACAGATTGTC-3' and a PstI-tagged reverse primer 5'-tatagacgtcCTGTTAAGCCTGAAGCCCCAAGA-3'. The PCR product was cloned downstream of the isopropyl-1-thio--D-galactopyranoside-inducible P_TAC promoter into the BamHI and PstI sites of the bacterial expression vector pMAL-C2X, to express the TMLH as a fusion protein with maltose-binding protein. The ORF was sequenced to exclude sequence errors introduced by PCR after which the construct was transformed into the Escherichia coli strain BL21. Transformed cells were grown in LB-medium with 100 µg/ml ampicillin to an A₆₀₀ of 0.7 and isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM to induce fusion protein expression. After 2 h, cells were pelleted and lysed in 1/10 of the culture volume in a 10 mM sodium phosphate buffer, pH 7.4, containing 140 mM NaCl by sonicating two times for 15 s at 8 watts. The bacterial lysate was centrifuged for 10 min at 14,000 × g, and the pellet was discarded. Fusion proteins were purified from the supernatant following the specifications of the manufacturer (New England BioLabs) and stored at 20 °C. This fusion protein was used to raise an antiserum in a rabbit as described earlier (29).

Density Gradient Analysis-- Kidneys were obtained from male Wistar rats and homogenized in 5 mM MOPS buffer, pH 7.4, containing 250 mM sucrose and 2 mM EDTA. A postnuclear supernatant was produced by centrifugation of the homogenate at 600 × g for 10 min at 4 °C and subfractionated by equilibrium density gradient centrifugation in a linear Nycodenz gradient as described previous (30). Glutamate dehydrogenase, catalase, -hexosaminidase, phosphogluco isomerase, were used as markers for mitochondria, peroxisomes, endoplasmic reticulum, and cytosol, respectively. The activity of the marker enzymes was determined as described previously (31, 32).

Immunoblot Analysis-- A Multiphor II Nova Blot electrophoretic transfer unit (Amersham Pharmacia Biotech) was used to transfer proteins onto a Nitrocellulose sheet (Schleicher & Schuell, Dassel, Germany) as described by the manufacturer. After blocking of nonspecific binding sites with 50 g/liter Protifar and 10 g/liter bovine serum albumin in 1 g/liter Tween-20/phosphate-buffered saline for 1 h, the blot was incubated for 2 h with a 1:200 dilution of rabbit polyclonal antibodies raised against human recombinant TMLH fused to maltose-binding protein (purified as described above) in the same buffer without Protifar. Goat anti-rabbit IgG antibodies conjugated to alkaline phosphatase were used for detection, according to the manufacturer's instructions (Bio-Rad).

Gel Filtration and Blue Native PAGE-- For gel filtration analysis a Superdex 200 column (Amersham Pharmacia Biotech) was used. A 20 mM ethanolamine buffer, pH 9.3, containing 100 g/liter glycerol, 2 mM sodium ascorbate, and 5 mM DTT was used as eluant at a flow rate of 0.4 ml/min. All analyses were performed at 4 °C. The column was calibrated under identical conditions with the following protein standards: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (14 kDa), all from Amersham Pharmacia Biotech. The ¹⁰log of the molecular mass of the protein standards was plotted against the corresponding elution fractions, and the molecular mass of TMLH was calculated by interpolation.

Blue native PAGE was performed as described previously using a 6-14% polyacrylamide gradient gel (33, 34). Citrate synthase (87 kDa) from pig heart (Sigma) and bovine serum albumin (66 kDa) were used as protein standards.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of TMLH from Rat Kidney-- Because kidney contains the highest TMLH activity in the rat (35), this tissue was used as source of enzyme for the purification of TMLH using liquid chromatography. An overview of the purification scheme is given in Table I. TMLH activity was retained completely by all columns used and eluted as a single peak during all purification steps. The presence of ascorbate was essential for preserving the enzymatic activity during the later purification steps and subsequent storage at 80 °C. Samples obtained after each purification step were analyzed by SDS-PAGE followed by silver staining (Fig. 1). A single protein band with an apparent molecular mass of 43 kDa was observed after the last purification step.

View larger version (84K): [in this window] [in a new window]   Fig. 1.   Overview of TMLH purification. Protein samples of the various purification steps were analyzed by 12% SDS-PAGE followed by silver staining. Lane 1, molecular mass marker; lane 2, 20,000 × g rat kidney supernatant, lane 3, low salt precipitate and pooled fractions of Q-Sepharose (lane 4), Butyl-Sepharose (lane 5), and hydroxylapatite CHT-II (lane 6).

Identification of the cDNA Encoding TMLH-- Attempts to directly sequence the protein by Edman degradation failed, suggesting that the N terminus of TMLH is blocked. Therefore, the purified protein was digested with trypsin and analyzed by MALDI-TOF MS. Because no match was found in the non-redundant data base (Swiss-Prot/TREMBL), two peptides were selected for sequencing by Q-TOF MS, which resulted in the following sequences: TLLVDGFYAAQQVLQR (1821.99 Da) and MWYFTSDFRS (1339.58 Da). When the non-redundant data base was searched with these peptides sequences, both showed high homology with the human hypothetical protein FLJ10727 (GenBank^TM accession number: NP_060666). Subsequent searches in the EST (Expressed Sequence Tag) data base identified several rat, mouse, and human EST clones with high homology to the peptide sequences. The homologous human ESTs all corresponded to the FLJ10727 cDNA (GenBank^TM accession number: AK001589). Interestingly, the translated FLJ10727 cDNA showed high homology with human, rat, and Pseudomonas sp. AK-1 -butyrobetaine hydroxylase. Based on the EST data, primers were selected to amplify the ORFs from rat, human, and mouse kidney cDNA. The rat and mouse amplicons both contained an ORF of 1218 base pairs, coding for a polypeptide of 405 amino acids with a predicted molecular mass of 47.5 kDa (GenBank^TM accession numbers: AF374406 and AY033513, respectively). When the theoretical trypsin digest of the translated rat ORF was compared with the MALDI-TOF MS spectrum of the purified TMLH, 12 of the 27 theoretical peptides could be matched, which corresponds with a protein coverage of 34%.

The human amplicon contained an ORF of 1266 base pairs, coding for a polypeptide of 421 amino acids with a predicted molecular mass of 49.5 kDa (GenBank^TM accession number: AF373407). The human TMLH cDNA sequence was identical to the FLJ10727 cDNA. This sequence is derived from genomic clone NT025307, which has been mapped to Xq28. BLASTn analysis of the human genome data base using the human TMLH cDNA as query showed that the TMLH gene spans about 130 kb and consists of at least eight exons.

The translated ORFs of rat and mouse both have 88% positional identity with the human TMLH protein. The rat and mouse proteins are also highly homologous and share 92% positional identity.

Expression of the Rat and Human TMLH cDNA-- When the putative rat and human TMLH cDNAs were expressed in either E. coli (as maltose-binding fusion protein) or S. cerevisiae, no TMLH activity could be detected in lysates of these cells. Therefore, both ORFs were cloned into the eukaryotic expression vector pcDNA3 and transiently transfected to COS cells. As a negative control, the pcDNA3 vector without insert was included in the transfection experiment. After 48 h, TMLH activity was measured in the lysates of the transfected cells employing the TMLH assay, which is based on the measurement of hydroxytrimethyllysine by HPLC tandem MS. Incubations were performed both in the presence and absence of substrate to show that the formation of hydroxytrimethyllysine was trimethyllysine-dependent. High TMLH activity could be measured in lysates of COS cells transfected with the putative rat and human TMLH cDNA, whereas only low (endogenous) TMLH activity was measured in lysates of cells transfected with the pcDNA3 vector without insert (Fig. 2A).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Transient transfection of rat and human TMLH cDNA in COS cells. A, P = pcDNA3 without insert, R = pcDNA3 + rat ORF and H = pcDNA3 + human ORF. Hydroxytrimethyllysine formation in incubations with (+) or without () addition of trimethyllysine, respectively. Transfections were performed in triplicate. B, immunoblot analysis of COS cell lysates with TMLH antibody. COS cells transfected with: pcDNA3 without insert (lane 1), pcDNA3 + rat ORF (lane 2), pcDNA3 + human ORF (lane 3). Lane 4 contains purified rat TMLH.

Subsequent immunoblot analysis using the TMLH antibody showed a band with the same molecular mass as the purified TMLH in cells transfected with the rat and human ORF, which was hardly detectable in lysates of cells transfected with pcDNA3 without insert. Additionally, the amount of immunoreactive material was proportional to the TMLH activity (Fig. 2B).

Subcellular Localization of TMLH-- To investigate the subcellular localization of TMLH, a density gradient analysis was performed with rat kidney homogenate. All the TMLH activity was associated with the particulate fraction, which was loaded on a Nycodenz density gradient. The activity profile in the gradient exactly coincided with that of the mitochondrial marker glutamate dehydrogenase, confirming the mitochondrial localization of TMLH (Fig. 3). When we analyzed the gradient-fractions by immunoblot analysis using antibodies raised against recombinant TMLH, the pattern of the immunoreactive material corresponded exactly with the TMLH activity profile (Fig. 3).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Subcellular localization of TMLH in rat kidney by density gradient analysis. A, marker enzymes: glutamate dehydrogenase (mitochondria, ), catalase (peroxisomes, ), -hexosaminidase (endoplasmic reticulum, ), phosphogluco isomerase (cytosol, ×). The highest activity in a particular fraction was set at 100%. B, TMLH activity. C, immunoblot analysis of gradient fractions with TMLH antibody; H = homogenate, P = particulate fraction, and S = supernatant.

Processing of TMLH-- With a calculated molecular mass of 47.5 kDa, the size of the translated ORF of rat TMLH is not in agreement with that of the purified protein, which has an apparent molecular mass of 43 kDa. Additionally, the COS cell transfection experiment showed that the produced human and rat TMLH both have the same apparent molecular mass as the purified rat TMLH, although the calculated molecular masses are 47.5 and 49 kDa, respectively. The 5'-end of the rat cDNA, as well as the mouse and human cDNAs, contained a second putative start codon. The use of this methionine would result in a protein of 42 kDa, and we therefore expressed the shorter rat protein in S. cerevisiae to investigate whether translation starts at this methionine. Immunoblot analysis of the yeast lysate with the TMLH antibody, however, clearly showed that the size of the expressed protein is smaller than the purified rat TMLH, indicating that this methionine is not used as start codon (Fig. 4). Another possibility is that TMLH is synthesized as a 47.5-kDa precursor, which is processed after import into the mitochondrion. The protein sequences of rat, mouse, and human TMLH indeed contain a putative N-terminal mitochondrial targeting sequence as determined by the Predotar version 0.5 prediction program.² Immunoblot experiments support this hypothesis, because expression of the full-length rat TMLH in S. cerevisiae resulted in a protein of 47.5 kDa (the predicted molecular mass of the translated rat ORF), but also showed a band with the same molecular mass as the purified rat protein (Fig. 4). Together, these results suggest that a 47.5-kDa precursor protein is synthesized and subsequently processed between the first and second methionine, resulting in a mature protein of ~43 kDa.

View larger version (88K): [in this window] [in a new window]   Fig. 4.   Expression of rat TMLH in S. cerevisiae. Immunoblot analysis of yeast lysates of cells transformed with pYES2 without insert (lane 1), pYES2 + rat TMLH (lane 2), pYES2 + rat TMLH starting from the second start codon (lane 3). Lane 4 contains purified rat TMLH.

Characterization of the Purified TMLH-- The enzyme has a broad pH optimum between 6.5 and 7.5 at 37 °C, which is in agreement with previous results (20). K_m values of trimethyllysine, -ketoglutarate, and Fe²⁺ were determined for the highly purified enzyme from Lineweaver-Burk double-reciprocal plots and were 1.1 mM, 109 µM, and 54 µM, respectively (Fig. 5). The K_m value of trimethyllysine is in agreement with the results of Sachan et al. (21), who determined a K_m value of 1.6 mM for the partially purified rat liver enzyme. Two other groups have determined K_m values of 0.1 mM (20) and 0.13 mM (36) for the rat and bovine liver enzymes, respectively, which are considerably lower than the K_m value determined in this study. The K_m values found previously for -ketoglutarate (480 and 220 µM) and Fe²⁺ (21 and 60 µM) are in agreement with our results (20, 36).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Km curve and Lineweaver-Burke plot for (A) trimethyllysine, (B) -ketoglutarate, and (C) Fe2+.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Native molecular mass determination of TMLH by gel filtration analysis and blue native PAGE. A, log molecular mass versus elution fraction of proteins standards () and TMLH (). The molecular mass of the standards are: thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; chymotrypsinogen A, 25 kDa; and ribonuclease A, 14 kDa. B, blue native PAGE gel loaded with; lane 1, citrate synthase (87 kDa); lane 2, TMLH; and lane 3, bovine serum albumin (66 kDa).

Native Molecular Mass Determination of Purified TMLH-- Gel filtration analysis showed that the native enzyme has a molecular mass of ~87 kDa, suggesting that TMLH has a dimeric configuration (Fig. 6A). This result was supported by blue native PAGE analysis (Fig. 6B), which showed that TMLH has a similar size as citrate synthase (87 kDa). MALDI-TOF analysis demonstrated that the protein band of ~87 kDa only contained TMLH, suggesting that TMLH is a homodimer.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To identify the genes encoding the enzymes of the carnitine biosynthetic pathway we previously purified rat liver -trimethylaminobutyraldehyde dehydrogenase and -butyrobetaine hydroxylase, the penultimate and ultimate enzymes in carnitine biosynthesis, respectively. We used protein sequence data in combination with EST data base searching to identify the corresponding rat and human cDNAs (16, 17). In this study the same approach was used to identify TMLH, which mediates the first step in carnitine biosynthesis. The enzyme was purified from rat kidney to near homogeneity and used for peptide sequencing. Subsequently, the resulting peptide sequences were used to search the EST data base, and ORFs were identified from rat, mouse, and human origin with high homology to the peptide sequences. The following observations demonstrated that the identified rat cDNA truly encodes TMLH. First, the peptide sequences obtained from the purified kidney TMLH exactly matched two stretches of sequence from the translated coding region of the rat cDNA. Second, the heterologously expressed rat cDNA exhibited high TMLH activity. Third, the peptide pattern of the purified rat TMLH determined by MALDI-TOF analysis matched the theoretical trypsin digest of the translated rat ORF. Finally, the antibody raised against recombinant TMLH recognized the purified enzyme.

Because the human homologue of the rat TMLH has 88% positional identity with the rat protein and exhibited high TMLH activity in the heterologous expression system, the corresponding cDNA encodes human TMLH. Data base searching showed that the TMLH cDNA is identical to the FLJ10727 cDNA and that the TMLH gene is localized at Xq28. Although we did not express the mouse ORF, it has 92% positional identity with the rat TMLH, and therefore most likely represents the mouse homologue of TMLH.

Purified TMLH behaves as an 87-kDa enzyme in both gel filtration and blue native PAGE analysis. Because a single protein of 43 kDa was present in the final purification sample and the MALDI-TOF analysis of the blue native PAGE sample demonstrated that the dimer consisted of a single protein, TMLH appears to be homodimer. The last enzyme of carnitine biosynthesis, -butyrobetaine hydroxylase, has considerable homology with TMLH and has also been reported to function as a homodimer (37-39). Analysis of the non-redundant data base with the BLASTp algorithm using rat TMLH as query, only retrieved -butyrobetaine hydroxylase sequences from several organisms. No homology was found with other -ketoglutarate-dependent dioxygenases, suggesting that TMLH and -butyrobetaine hydroxylase belong to a separate subclass of dioxygenases.

TMLH has been reported to be localized in mitochondria (8, 21), although this conclusion was drawn from relatively crude experiments involving differential centrifugation. Therefore, the subcellular localization of TMLH in rat kidney was re-investigated by subcellular fractionation using density gradient analysis. The TMLH activity profile and the distribution of immunoreactive material clearly showed that TMLH is localized exclusively in mitochondria. The expression studies of TMLH in S. cerevisiae suggest that translation of TMLH starts at the first available start codon, which results in the formation of a 47.5-kDa precursor protein. This precursor is subsequently processed to the mature 43-kDa protein, presumably upon import into mitochondria where the mitochondrial import machinery removes the N-terminal presequence.

The mitochondrial localization of TMLH is remarkable, because the other three enzymes of the carnitine biosynthesis are localized in the cytosol. The submitochondrial localization of TMLH will have implications for the substrate flow and regulation of the carnitine biosynthesis. If TMLH is localized in the mitochondrial matrix, the existence of transport system to shuttle substrate and product over the inner mitochondrial membrane would be required. In contrast, if TMLH is present in either the inner membrane space or the outer mitochondrial membrane, no transport system would be needed because the outer mitochondrial membrane is permeable for small molecules.

	ACKNOWLEDGEMENTS

We gratefully acknowledge S. van Woerkom for providing the rat kidneys, D. Speijer for the MALDI-TOF analysis, and H. R. Waterham for critical reading of the manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF374406, AY033513, and AF373407.

^¶ To whom correspondence should be addressed: Depts. of Clinical Chemistry and Pediatrics, University of Amsterdam, Academic Medical Center, Laboratory for Genetic Metabolic Diseases (F0-224), P. O. Box 22700, Amsterdam 1100 DE, The Netherlands. Tel.: 31-20-566-5958; Fax: 31-20-696-2596; E-mail: wanders@amc.uva.nl.

Published, JBC Papers in Press, June 28, 2001, DOI 10.1074/jbc.M105929200

² Available at www.inra.fr/Internet/Produits/Predotar.

	ABBREVIATIONS

The abbreviations used are: TMLH, -trimethyllysine hydroxylase; MS, mass spectrometry; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption time of flight; Q-TOF, quadrupole-time-of-flight; MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; ESI, electrospray ionization; CMV, cytomegalovirus; EST, expressed sequence tag; kb, kilobase(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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