Abstract
epsilon-N-Trimethyllysine hydroxylase (EC ) is the first enzyme in the biosynthetic pathway of l-carnitine and catalyzes the formation of beta-hydroxy-N-epsilon-trimethyllysine from epsilon-N-trimethyllysine, a reaction dependent on alpha-ketoglutarate, Fe(2+), 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 epsilon-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, alpha-ketoglutarate, and Fe(2+) were 1.1 mm, 109 microm, and 54 microm, 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 epsilon-N-trimethyllysine hydroxylase is a homodimer.
Highlights
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]
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]
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
Summary
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). 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. Bound proteins were eluted with a linear gradient from 1 M KCl ϩ 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 ϭ 2 cm; h ϭ 5 cm; flow: 1 ml/min) equilibrated with the same buffer. The PCR product was cloned downstream of the isopropyl-1-thio--D-galactopyranoside-inducible PTAC promoter into the BamHI and PstI sites of TMLH in Carnitine Biosynthesis
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