Expression and characterization of the human mitochondrial leucyl-tRNA synthetase

A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeu RS) has been identified from the EST databases. Analysis of the protein enc oded by this cDNA indicates that the protein is 903 amino acids in length a nd contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contain s the characteristic motifs of class I aminoacyl-tRNA synthetases and regio ns of high homology to other mitochondrial and bacterial LeuRS proteins. Th e mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa, The human mtLeuRS is cap able of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K-M and k(cat) values for ATP:PPi exchange and for the aminoacylation reaction have been determined. (C) 2000 Elsevier Science B.V. All rights reserved.