Regulation of photosynthetic GAPDH dissected by mutants.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which is part of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependent activity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A2B2, A8B8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A2B2-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A8B8, B8). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutant A(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulation principally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate or NADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redox condition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity, both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and current functional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a conformational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latter residue is thus distracted from binding the 2'-phosphate of NADP, with the final effect that the enzyme relaxes to a conformation leading to a slower NADPH-dependent catalytic activity.
Sparla
Francesca
F
Laboratory of Molecular Plant Physiology, Department of Biology, University of Bologna, 40126 Bologna, Italy.
Zaffagnini
Mirko
M
Wedel
Norbert
N
Scheibe
Renate
R
Pupillo
Paolo
P
Trost
Paolo
P
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D007527:Isoenzymes; D009154:Mutation; D030781:Organisms, Genetically Modified; D010084:Oxidation-Reduction; D010788:Photosynthesis; D010940:Plant Proteins / Q000378:metabolism; D021122:Protein Subunits; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology*
D007527:Isoenzymes; D010940:Plant Proteins; D021122:Protein Subunits; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases