The in-vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts

Abstract

Full-length cDNA clones encoding shikimate kinase (EC 2.7.1.71), an enzyme of the central section of the shikimate pathway, have been isolated from tomato (Lycopersicon esculentum L., cv. UC82b). The open reading frame has the capacity to encode a peptide of 300 amino acids. The in-vitro synthesized peptide catalysed the phosphorylation of shikimate thus confirming the identity of the isolated cDNA clones. The N-terminal portion of the deduced amino acid sequence resembles known chloroplast-specific transit peptides. The existence of such a transit peptide was proven by the uptake of the in-vitro synthesized peptide as well as its processing by isolated chloroplasts. Multiple sites of polyadenylation were observed in shikimate kinase mRNAs. The results of Northern and Southern blot analyses are consistent with the existence of only one shikimate kinase gene per haploid genome in tomato. These results are discussed with respect to the dual pathway hypothesis of the shikimate pathway in higher plants.