Minisymposium 12: Photosynthetic Carbon Metabolism

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Abs # M1201: Regulatory complexity of photosynthetic glyceraldehyde-3-phosphate dehydrogenase

Presenter:	Sparla, Francesca       Contact Presenter
Authors	Sparla, Francesca  (A)   Fermani, Simona  (B)   Zaffagnini, Mirko  (A)   Marri, Lucia  (A)   Falini, Giuseppe  (B)   Pupillo, Paolo  (A)   Trost, Paolo  (A)
Affiliations:	(A): Department of Evolutionary Experimental Biology, University of Bologna (B): Department of Chemistry "G. Ciamician", University of Bologna
Web Site:	http://www.dipartimentobiologia.it/research/rupupillo.asp

The Calvin cycle includes a single reductive step catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In higher plants, two distinct isoforms of GAPDH are present in chloroplasts: a major heterotetrameric isozyme (A₂B₂-GAPDH) and a less abundant homotetrameric isozyme (A₄-GAPDH). Both enzymes are regulated by light via thioredoxins and metabolites through mechanisms which display both unique features and common traits. In A₂B₂-GAPDH, the C-terminal extension (CTE) of B-subunits is responsible for the thioredoxin-mediated regulation. After formation of a disulfide within the CTE, this protruding regulatory domain is proposed to fold nearby the active site of the protein and interact with essential residues for coenzyme recognition and catalysis. As a consequence, the NADPH-dependent activity of the enzyme is specifically slowed down (Sparla et al., J Biol Chem, 2002; Plant Physiol, 2005), consistent with the down regulation of the Calvin cycle in the dark, when chloroplast thioredoxins are relatively more oxidized. Mechanistic details of CTE-dependent A₂B₂-GAPDH regulation are now revealed by the crystallographic structure of oxidized A₂B₂-GAPDH combined with mutant analysis. Although A₄-GAPDH does not contain CTE, it responds to thioredoxins and metabolites via the small regulatory peptide CP12. The CTE of B-subunits and the C-terminal portion of CP12 have similar sequences, possibly reflecting a common evolutionary origin and suggesting a similar regulatory function down to the molecular level. Through the action of CP12, a supramolecular complex involving GAPDH and phosphoribulokinase (PRK) is assembled. Formation of the A₄-GAPDH/CP12/PRK complex leads to a strong down regulation of both enzyme activities, thereby providing an alternative regulatory mechanism of the Calvin cycle (Marri et al., Plant Physiol, 2005). Dissociation of the A₄-GAPDH/CP12/PRK complex in vitro occurs under distinct conditions, including treatments with reduced thioredoxin f or metabolites (BPGA, NADP, ATP). Indeed A₄-GAPDH, previously considered as a constitutively active GAPDH isoform, acquires remarkable regulatory properties thanks to the interaction with CP12 and PRK. We thus postulate that during dark to light transition the reduction of thioredoxins by photosystem I and the increase of Calvin cycle intermediates lead to complex dissociation and consequent enzyme re-activation.