Expression and characterization of a human mitochondrial phenylalanyl-tRNAsynthetase

Human mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) has been identif ied from the human EST database. Using consensus sequences derived from con served regions of the alpha and beta-subunits from bacterial PheRS, two par tially sequenced cDNA clones were identified. Unexpectedly, sequence analys is indicated that one of these clones was a truncated form of the other. De tailed analysis indicates that unlike the (alpha beta)(2) structure of the prokaryotic and eukaryotic cytoplasmic forms of PheRS, the human mtPheRS co nsists of a single polypeptide chain. This protein has been cloned and expr essed in Escherichia coli. Gel filtration and analytical velocity sedimenta tion centrifugation indicate that the human mtPheRS is active in a monomeri c form. The N-terminal 314 amino acid residues appear to be analogous to th e alpha-subunit of the prokaryotic PheRS, while the C-terminal 100 amino ac id residues correspond to a region of the beta-subunit known to interact wi th the anticodon of tRNA(Phe). Comparisons with the sequences of PheRS from yeast and Drosophila mitochondria indicate they are 42% and 51% identical with the human mtPheRS, respectively. Sequence analysis confirms the presen ce of motifs characteristic of class II aminoacyl-tRNA synthetases. K-M and k(cat) values for ATP:PPi exchange and for the aminoacylation reaction car ried out by human mtPheRS have been determined. Evolutionary origins of thi s small monomeric human mtPheRS are unknown, however, implications are that this enzyme is a result of the simplification of the more complex (alpha b eta)(2) bacterial PheRS in which specific functional regions were retained. (C) 1999 Academic Press.