Light-dependent Activation of NADP-Malate Dehydrogenase: a Complex Process

Abstract

After the discovery of the light-activation properties of the chloroplastic NADP-dependent malate dehydrogenase in 1970, the elements of the activation pathway were identified and shown to consist of the stromal proteins of the ferredoxin/thioredoxin system. It was further demonstrated that the activation was a reductive process during which disulfides were reduced into dithiols by reduced thioredoxin. Sequence alignments with the permanently active NAD-malate dehydrogenases revealed N- and C-terminal extensions specific for the light-regulated form. A regulatory disulfide was identified in the amino-terminal extension by chemical derivatisation: its reduction was correlated to the activation of the enzyme. The use of site-directed mutagenesis techniques revealed the complexity of the intramolecular activation mechanism, showing that two different disulfides were reduced per subunit of this homodimeric enzyme: one located in the N-terminal extension, the other in the C-terminal extension. A model was proposed where the C-terminal extension locks the access to the active site, whereas the N-terminal extension governs the conformation of the active site. The identification of the catalytic histidine allowed us to test the accessibility of the active site and to demonstrate the validity of the proposed model.

Thioredoxins: structure and function in plant cells

“…and this can be achieved only through thioredoxin (either wz or / activates this enzyme with the same efficiency under certain conditions). The enzyme has been isolated from a variety of plant sources including C4 plants (sorghum, corn) and C3 plants (pea, spinach) (for a very recent review on this protein, see Miginiac-Maslow et al, 1997). NADP-MDH is a dimeric enzyme constituted of two identical subunits with a molecular weight of c. 40 kDa (Metzler, Rothermel & Nelson, 1989, Cretin et al, 1990, Agostino, Jeffery & Hatch, 1992.…”

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

“…The regulatory aspects of the light-dependent chloroplast enzymes have been quite extensively studied (for reviews see Buchanan 1992;Wolosiuk et al 1993;Miginiac-Maslow et al 1997); however, structural information has not been available. During the past decade, the structures of the members of the ferredoxin/thioredoxin system and of two well-studied target enzymes have been solved.…”

Redox Regulation of Arabidopsis 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase

“…In chloroplasts, four enzymes of the BensonCalvin cycle, spinach (Spinacia oleracea) phosphoribulokinase (Brandes et al, 1996), pea (Pisum sativum) glyceraldehyde-3-phosphate dehydrogenase (Li and Anderson, 1997) and Fru-1,6-bisphosphate phosphatase (Jacquot et al, 1995), and Arabidopsis sedoheptulose-1,7-bisphosphate phosphatase (Willingham et al, 1994) and the larger of two isoforms of Rubisco activase (Zhang and Portis, 1999), a H ϩ -ATPase (Schwarz et al, 1997), and NADP-malate dehydrogenase (Miginiac-Maslow et al, 1997;Ocheretina et al, 2000) are regulated by light via the ferredoxin/thioredoxin (Fd/TRX) system (Buchanan et al, 1994;Ruelland and Miginiac-Maslow, 1999;Schü rmann and Jacquot, 2000). In this paper, we show that Arabidopsis DAHP synthase requires reduced TRX for enzyme activity, thereby linking carbon flow into the shikimate pathway to electron flow from photosystem I.…”