The cDNA sequences encoding cytosolic and lightmodulated plastidic glucose-6-phosphate dehydrogenase (G6PDH) from potato were modified by polymerase chain reaction and subsequently overexpressed in Escherichia coli. Characterization of the recombinant enzymes showed that they closely resembled their native counterparts. Treatment with reduced dithiothreitol or glutathione led to inactivation of plastidic G6PDH, whereas the activity of the cytosolic isoenzyme was not influenced by reduction. As for the native enzyme, inactivation of recombinant plastidic G6PDH was accelerated by thioredoxin m and could be fully reversed by subsequent addition of oxidant. To identify the residues which are involved in redox regulation of plastidic G6PDH, each of the six cysteines in the mature protein sequence was exchanged separately for serine by sitedirected mutagenesis. Two mutant proteins exhibited characteristics of the reduced wild-type enzyme. Exchange of either Cys 149 or Cys 157 to serine abolished the regulatory properties, suggesting that these cysteine residues are the sites responsible for redox-mediated inactivation of plastidic G6PDH. G6PDH1 (EC 1.1.1.49) catalyzes the first step of the oxidative pentose-phosphate pathway. The main function of the enzyme is to provide NADPH for reductive biosyntheses. In plant tissues, at least two G6PDH isoforms exist in two different compartments, one in the cytosol and one in the chloroplast stroma (1, 2).The activity of several chloroplast enzymes is known to be regulated by reversible thiol-disulfide interchange (3). During photosynthetic electron transport in the light, covalent redox modification mediated by a redox chain (the ferredoxin-thioredoxin system) leads to reductive light activation of several stromal target enzymes, e.g. fructose-1,6-bisphosphatase, NADP-malate dehydrogenase, phosphoribulokinase, and others (4). In contrast, chloroplast G6PDH is inactivated in the light (5) or by reductants (6, 7) and is therefore active in the oxidized state. This regulation prevents futile cycling, i.e. simultaneous carbohydrate synthesis in the Calvin cycle and catabolism by the oxidative pentose-phosphate pathway. Thus, in accordance with its physiological role in chloroplasts, G6PDH is active only during the dark phase, when NADPH supply by the photosynthetic electron flow ceases.Recently, we isolated cDNA sequences encoding cytosolic and plastidic G6PDH from potato (8, 9). Both plant isoforms contain six cysteine residues, but none of them at conserved positions. Notably, in the plastidic sequence all cysteines are located within a relatively short amino-terminal stretch of about 100 amino acids (9) within the NADP binding domain (10). Comparison of the deduced amino acid sequences with those of the redox-modulated G6PDH from cyanobacteria (11-13) revealed substantial differences in the primary structures. The cyanobacterial G6PDH sequences contain two conserved cysteines at completely different positions compared with plastidic G6PDH.To locate the cysteine residues involved...

SummaryWe describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox-regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C-terminal Strep tag, the enzyme retained G6PDH activity upon af®nity puri®cation. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein-mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The K m values determined for both substrates were signi®cantly lower compared to those of P1. The high V max and K i [NADPH] values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid-localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features.

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We report on the isolation and characterization of full-length cDNA sequences coding for N-acetylglucosaminyltransferase I (GnTI) from potato (Solanum tuberosum L.), tobacco (Nicotiana tabacum L.), and Arabidopsis. The deduced polypeptide sequences show highest homology among the solanaceous species (93% identity between potato and tobacco compared with about 75% with Arabidopsis) but share only weak homology with human GnTI (35% identity). In contrast to the corresponding enzymes from animals, all plant GnTI sequences identified are characterized by a much shorter hydrophobic membrane anchor and contain one putative N-glycosylation site that is conserved in potato and tobacco, but differs in Arabidopsis. Southern-blot analyses revealed that GntI behaves as a single-copy gene. Northern-blot analyses showed that GntI-mRNA expression is largely constitutive. Arabidopsis cgl mutants deficient in GnTI activity also possess GntI mRNA, indicating that they result from point mutations. GntI-expression constructs were tested for the ability to relieve the GnTI block in protoplasts of the Arabidopsis cgl mutant and used to obtain transgenic potato and tobacco plants that display a substantial reduction of complex glycan patterns. The latter observation indicates that production of heterologous glycoproteins with little or no antigenic glycans can be achieved in whole plants, and not in just Arabidopsis, using antisense technology.

In a recent paper (Wenderoth et al., J Biol Chem 272: 26985-26990, 1997) we reported that the positions of the two redox regulatory cysteines identified in a plastidic G6PD isoform from potato (Solanum tuberosum L.) differ substantially from those conserved in cyanobacterial G6PDH sequences. To investigate the origin of redox regulation in G6PDH enzymes from photoautotrophic organisms, we isolated and characterized several G6PD cDNA sequences from higher plants and from a green and a red alga. Alignments of the deduced amino acid sequences showed that the cysteine residues cluster in the coenzyme-binding domain of the plastidic isoforms and are conserved at three out of six positions. Comparison of the mature proteins and the signal peptides revealed that two different plastidic G6PDH classes (P1 and P2) evolved from a common ancestral gene. The two algal sequences branch off prior to this class separation in higher plants, sharing about similar amino acid identity with either of the two plastidic G6PDH classes. The genes for cytosolic plant isoforms clearly share a common ancestor with animal and fungal G6PDH homologues, whereas the cyanobacterial isoforms branch within the eubacterial G6PDH sequences. The data suggest that cysteine-mediated redox regulation arose independently in G6PDH isoenzymes of eubacterial and eukaryotic lineages.