title: Functional analysis of TMLH variants and definition of domains required for catalytic activity and mitochondrial targeting.
abstract: epsilon-N-Trimethyllysine hydroxylase (TMLH) (EC 1.14.11.8) is a non-heme-ferrous iron hydroxylase, Fe(++) and 2-oxoglutarate (2OG) dependent, catalyzing the first of four enzymatic reactions of the highly conserved carnitine biosynthetic pathway. Otherwise from all the other enzymes of carnitine biosynthesis, TMLH was found to be associated to the mitochondrial fraction. We here report molecular cloning of two alternative spliced forms of TMLH, which appear ubiquitously expressed in human adult and fetal tissues. The deduced proteins are designated TMLH-a and TMLH-b, and contain 421 and 399 amino acids, respectively. They share the first N-terminal 332 amino acids, including a mitochondrial targeting signal, but diverge at the C-terminal end. TMLH-a and TMLH-b exogenous expression in COS-1 cells shows that the first 15 amino acids are necessary and sufficient for mitochondrial import. Furthermore, comparative evolutionary analysis of the C-terminal portion of TMLH-a identifies a conserved domain characterized by a key triad of residues, His242-Glu244-His389 predicted to bind 2OG end. This sequence is conserved in the TMLH enzyme from all species but is partially substituted by a unique sequence in the TMLH-b variant. Indeed, TMLH-b is not functional by itself as well as a TMLH-H389L mutant produced by site directed mutagenesis. As great interest, we found that TMLH-b and TMLH-H389L, individually co-expressed with TMLH-a in COS-1 cells, negatively affect TMLH activity. Therefore, our studies on the TMLH alternative form provide relevant novel information, first that the C-terminal region of TMLH contains the main determinants for its enzymatic activity including a key H389 residue, and second that TMLH-b could act as a crucial physiological negative regulator of TMLH.
authors.affiliation: Institute of Genetics and Biophysics, Adriano Buzzati-Traverso (CNR), Naples, Italy.
authors.forename: Jlenia
authors.initials: J
authors.lastname: Monfregola
authors.forename: Armando
authors.initials: A
authors.lastname: Cevenini
authors.forename: Antonio
authors.initials: A
authors.lastname: Terracciano
authors.forename: Naomi
authors.initials: N
authors.lastname: van Vlies
authors.forename: Salvatore
authors.initials: S
authors.lastname: Arbucci
authors.forename: Ronald J A
authors.initials: RJ
authors.lastname: Wanders
authors.forename: Michele
authors.initials: M
authors.lastname: D'Urso
authors.forename: Frédéric M
authors.initials: FM
authors.lastname: Vaz
authors.forename: Matilde Valeria
authors.initials: MV
authors.lastname: Ursini
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D014644:Genetic Variation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D006899:Mixed Function Oxygenases; C436318:epsilon-N-trimethyllysine hydroxylase