GENE STRUCTURE, EXPRESSION IN ESCHERICHIA-COLI AND BIOCHEMICAL-PROPERTIES OF THE NAD(-DEPENDENT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM PINUS-SYLVESTRIS CHLOROPLASTS())

Photosynthetic eukaryotes typically possess two distinct glyceraldehyd e-3-phosphate dehydrogenases, an NAD(+)-specific enzyme in the cytosol (GapC: EC 1.2.1.12) and an NADP(+)-dependent enzyme in the chloroplas t (GapAB: EC 1.2.1.13). The gymnosperm Pinus sylvestris is an exceptio n in that it is known to express a gene encoding a transit peptide-bea ring GapC-like subunit that is imported into chloroplasts (GapCp), but the enzymatic properties of this novel GAPDH have not been described from any source. We have expressed the mature GapCp subunit from Pinus in Escherichia coli and have characterized the active enzyme. GapCp h as a specific activity of 89 units per milligram and is strictly NAD()-dependent, showing no detectable activity with NADP(+). Values of th e apparent K-m for NAD(+) and glyceraldehyde-3-phosphate were determin ed as 62 and 344 mu M, respectively. The Pinus GapCp1 gene possesses 1 2 introns, two in the region encoding the transit peptide and ten in t he region encoding the mature subunit, all of which are found at posit ions strictly conserved across genes for higher plant GapC. A cDNA enc oding a homologue of GapCp was isolated from the heterosporous fern Ma rsilea quadrifolia, indicating that NAD(+)-dependent chloroplast GAPDH also occurs in other higher plants. (C) 1998 Elsevier Science B.V.