Regulation of photosynthetic GAPDH dissected by mutants

Autor(en): Sparla, F
Zaffagnini, M
Wedel, N
Scheibe, R 
Pupillo, P
Trost, P
Stichwörter: 3-PHOSPHATE DEHYDROGENASE; CALVIN CYCLE ACTIVITY; CHLOROPLAST GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE; CRYSTAL-STRUCTURE; LIGHT-ACTIVATION; NADP-MALATE DEHYDROGENASE; NICOTINAMIDE ADENINE-DINUCLEOTIDE; OXIDATION-REDUCTION PROPERTIES; Plant Sciences; PLASTIDIAL THIOREDOXINS; REDOX REGULATION
Erscheinungsdatum: 2005
Herausgeber: AMER SOC PLANT BIOLOGISTS
Journal: PLANT PHYSIOLOGY
Volumen: 138
Ausgabe: 4
Startseite: 2210
Seitenende: 2219
Zusammenfassung: 
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 ( A 2 B 2, A 8 B 8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A(2)B(2)-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A(8)B(8), B-8). 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.
ISSN: 00320889
DOI: 10.1104/pp.105.062117

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