Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC–SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A2B2)n-GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A4B4 and A8B8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphospho­glycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.

Unravelling the regulation pathway of photosynthetic AB-GAPDH / Marotta, Roberto; DEL GIUDICE, Alessandra; Gurrieri, Libero; Fanti, Silvia; Swuec, Paolo; Galantini, Luciano; Falini, Giuseppe; Trost, Paolo; Fermani, Simona; Sparla, Francesca. - In: ACTA CRYSTALLOGRAPHICA. SECTION D, STRUCTURAL BIOLOGY. - ISSN 2059-7983. - 78:11(2022), pp. 1399-1411. [10.1107/S2059798322010014]

Unravelling the regulation pathway of photosynthetic AB-GAPDH

Alessandra Del Giudice;Luciano Galantini;
2022

Abstract

Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC–SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A2B2)n-GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A4B4 and A8B8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphospho­glycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.
2022
photosynthesis; redox regulation; cryo-electron microscopy; small-angle X-ray scattering; photosynthetic glyceraldehyde 3-phosphate dehydrogenase; AB-GAPDH
01 Pubblicazione su rivista::01a Articolo in rivista
Unravelling the regulation pathway of photosynthetic AB-GAPDH / Marotta, Roberto; DEL GIUDICE, Alessandra; Gurrieri, Libero; Fanti, Silvia; Swuec, Paolo; Galantini, Luciano; Falini, Giuseppe; Trost, Paolo; Fermani, Simona; Sparla, Francesca. - In: ACTA CRYSTALLOGRAPHICA. SECTION D, STRUCTURAL BIOLOGY. - ISSN 2059-7983. - 78:11(2022), pp. 1399-1411. [10.1107/S2059798322010014]
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