Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3 .2.3) was isolated from tobacco leaves. The cDNA clone contained an open re ading frame encoding the protein of 501 amino acids with a calculated molec ular mass of 56,926Da, preceded by a putative mitochondrial targeting signa l consisting of 86 amino acid residues. In fact, GAL dehydrogenase was loca lized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco c ontains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding to bacco GAL dehydrogenase into a pET expression vector to overexpress this pr otein in Escherichia coli. The partially purified recombinant enzyme was us ed for comparative studies on the native enzymes from tobacco and other sou rces; its enzymatic properties were similar to those of other GAL dehydroge nases.