| First Author | Vaz FM | Year | 2001 |
| Journal | J Biol Chem | Volume | 276 |
| Issue | 36 | Pages | 33512-7 |
| PubMed ID | 11431483 | Mgi Jnum | J:76186 |
| Mgi Id | MGI:2178769 | Doi | 10.1074/jbc.M105929200 |
| Citation | Vaz FM, et al. (2001) Molecular and Biochemical Characterization of Rat epsilon -N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis. J Biol Chem 276(36):33512-7 |
| abstractText | 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. |
