e-N-Trimethyllysine hydroxylase (TMLH) (EC 1.14.11.8) is a non-heme-ferrous iron hydroxylase, Feþþ 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.

Functional analysis of TMLH variants and definition of domains required for catalytic activity and mitochondrial targeting / J., Monfregola; Cevenini, Armando; A., Terracciano; N. V., Vlies; S., Arbucci; R. J., Wanders; M., D'Urso; F. M., Vaz; M. V., Ursini. - In: JOURNAL OF CELLULAR PHYSIOLOGY. - ISSN 0021-9541. - STAMPA. - 204:3(2005), pp. 839-847. [10.1002/jcp.20332]

Functional analysis of TMLH variants and definition of domains required for catalytic activity and mitochondrial targeting

CEVENINI, Armando;
2005

Abstract

e-N-Trimethyllysine hydroxylase (TMLH) (EC 1.14.11.8) is a non-heme-ferrous iron hydroxylase, Feþþ 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.
2005
Functional analysis of TMLH variants and definition of domains required for catalytic activity and mitochondrial targeting / J., Monfregola; Cevenini, Armando; A., Terracciano; N. V., Vlies; S., Arbucci; R. J., Wanders; M., D'Urso; F. M., Vaz; M. V., Ursini. - In: JOURNAL OF CELLULAR PHYSIOLOGY. - ISSN 0021-9541. - STAMPA. - 204:3(2005), pp. 839-847. [10.1002/jcp.20332]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/344807
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