Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the regulatory peptide CP12 a supramolecular complex in Arabidopsis (Arabidopsis ...thaliana) that could be reconstituted in vitro using purified recombinant proteins. Both enzyme activities were strongly influenced by complex formation, providing an effective means for regulation of the Calvin cycle in vivo. PRK and CP12, but not GapA (A₄ isoform of GAPDH), are redox-sensitive proteins. PRK was reversibly inhibited by oxidation. CP12 has no enzymatic activity, but it changed conformation depending on redox conditions. GapA, a bispecific NAD(P)-dependent dehydrogenase, specifically formed a binary complex with oxidized CP12 when bound to NAD. PRK did not interact with either GapA or CP12 singly, but oxidized PRK could form with GapA/CP12 a stable ternary complex of about 640 kD (GapA/CP12/PRK). Exchanging NADP for NAD, reducing CP12, or reducing PRK were all conditions that prevented formation of the complex. Although GapA activity was little affected by CP12 alone, the NADPH-dependent activity of GapA embedded in the GapA/CP12/PRK complex was 80% inhibited in respect to the free enzyme. The NADH activity was unaffected. Upon binding to GapA/CP12, the activity of oxidized PRK dropped from 25% down to 2% the activity of the free reduced enzyme. The supramolecular complex was dissociated by reduced thioredoxins, NADP, 1,3-bisphosphoglycerate (BPGA), or ATP. The activity of GapA was only partially recovered after complex dissociation by thioredoxins, NADP, or ATP, and full GapA activation required BPGA. NADP, ATP, or BPGA partially activated PRK, but full recovery of PRK activity required thioredoxins. The reversible formation of the GapA/CP12/PRK supramolecular complex provides novel possibilities to finely regulate GapA ("non-regulatory" GAPDH isozyme) and PRK (thioredoxin sensitive) in a coordinated manner.
Calvin–Benson cycle regulation is getting complex Gurrieri, Libero; Fermani, Simona; Zaffagnini, Mirko ...
Trends in plant science,
September 2021, 2021-09-00, 20210901, Letnik:
26, Številka:
9
Journal Article
Recenzirano
Oxygenic phototrophs use the Calvin–Benson cycle to fix CO2 during photosynthesis. In the dark, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), two enzymes of the ...Calvin–Benson cycle, form an inactive complex with the regulatory protein CP12, mainly under the control of thioredoxins and pyridine nucleotides. In the light, complex dissociation allows GAPDH and PRK reactivation. The GAPDH/CP12/PRK complex is conserved from cyanobacteria to angiosperms and coexists in land plants with an autoassembling GAPDH that is analogously regulated. With the recently described 3D structures of PRK and GAPDH/CP12/PRK, the structural proteome of this ubiquitous regulatory system has been completed. This outcome opens a new avenue for understanding the regulatory potential of photosynthetic carbon fixation by laying the foundation for its knowledge-based manipulation.
Two enzymes of the Calvin–Benson cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), together with the regulatory protein CP12, can assemble into an inactive multimeric complex. With the recent characterization of the structures of free PRK and GAPDH/CP12/PRK ternary complexes, the hierarchical process of protein assembly can be described at molecular definition.CP12 complexes are conserved in oxygenic phototrophs, but land plants also contain an autoassembling GAPDH isoform, evolutionarily derived from CP12. Both types of complexes form in the dark and dissociate in light, mainly under the control of thioredoxins and pyridine nucleotides.CP12 is a major light/dark regulator of the Calvin–Benson cycle in cyanobacteria and contributes to the more sophisticated regulation of the cycle in land plants, where dark complexes may play an additional role in protecting enzymes from proteolysis.
In oxygenic photosynthetic organisms, the activities of two Calvin cycle enzymes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH and phosphoribulokinase, PRK) are regulated by CP12-mediated complex ...formation. The
Arabidopsis genome contains three genes encoding different CP12 isoforms (CP12-1, At2g47400; CP12-2, At3g62410 and CP12-3, At1g76560), all plastid-targeted, as demonstrated by localization in the chloroplast stroma of CP12 precursor sequences fused with the green fluorescence protein (GFP). The disorder predictor PONDR
® classified
Arabidopsis CP12s as largely disordered proteins, and circular dichroism spectra confirmed these predictions. Based on sequence similarity, 66 CP12s from different organisms were identified and clustered in six types, with CP12-1 and -2 grouping together with other largely disordered sequences (Type I), while a lower level of disorder was predicted within the cluster including CP12-3 (Type II).
The three
Arabidopsis CP12 isoforms were expressed as mature recombinant forms and purified to homogeneity. Redox titrations demonstrated that the four conserved cysteines of each CP12 isoform could form two internal disulfide bridges with different midpoint redox potentials (
E
m,7.9 −326
mV and −350
mV in both CP12-1 and CP12-2;
E
m,7.9 −332
mV and −373
mV in CP12-3). In agreement with their similar redox properties, all CP12 isoforms formed,
in vitro, a supramolecular complex with GAPDH and PRK, with comparable inhibitory effects on both enzyme activities. In order to test whether CP12 isoforms might have broader regulatory functions than regulating Calvin cycle enzymes, CP12 proteins were analyzed for their capacity to bind plastidial glycolytic GAPDH (GapCp). To this purpose, the mature form of
Arabidopsis GapCp2 was cloned, expressed in recombinant form and purified to homogeneity. However, contrary to expectations, no CP12 isoform was able to bind GapCp2 under any of the conditions tested.
Bionic composites are an emerging class of materials produced exploiting living organisms as reactors to include synthetic functional materials in their native and highly performing structures. In ...this work, single wall carboxylated carbon nanotubes (SWCNT-COOH) were incorporated within the roots of living plants of
Arabidopsis thaliana
. This biogenic synthetic route produced a bionic composite material made of root components and SWCNT-COOH. The synthesis was possible exploiting the transport processes existing in the plant roots. Scanning electrochemical microscopy (SECM) measurements showed that SWCNT-COOH entered the vascular bundles of
A. thaliana
roots localizing within xylem vessels. SWCNT-COOH preserved their electrical properties when embedded inside the root matrix, both at a microscopic level and a macroscopic level, and did not significantly affect the mechanical properties of
A. thaliana
roots.
The regulatory isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-activated enzyme constituted by subunits
GapA and GapB. The NADPH-dependent activity of regulatory GAPDH from ...spinach chloroplasts was affected by the redox potential
( E
m
,7.9 , â353 ± 11 mV) through the action of thioredoxin f. The redox dependence of recombinant GapB ( E
m
,7.9 , â347 ± 9 mV) was similar to native GAPDH, whereas GapA was essentially redox-insensitive. GapB mutants having one or two
C-terminal cysteines mutated into serines (C358S, C349S, C349S/C358S) were less redox-sensitive than GapB. Different mutants
with other cysteines substituted by serines (C18S, C274S, C285S) still showed strong redox regulation. Fully active GapB was
a tetramer of B-subunits, and, when incubated with NAD, it associated to a high molecular weight oligomer showing low NADPH-dependent
activity. The C-terminal GapB mutants (C358S, C349S, C349S/C358S) were active tetramers unable to aggregate to higher oligomers
in the presence of NAD, whereas other mutants (C18S, C274S, C285S) again behaved like GapB.
We conclude that a regulatory disulfide, between Cys-349 and Cys-358 of the C-terminal extension of GapB, does form in the
presence of oxidized thioredoxin. This covalent modification is required for the NAD-dependent association into higher oligomers
and inhibition of the NADPH-activity. By leading to GAPDH autoinhibition, thioredoxin and NAD may thus concur to the dark
inactivation of the enzyme in vivo .
Thioredoxins (TRXs) are major protein disulfide reductases of the cell. Their redox activity relies on a conserved Trp-Cys-(Gly/Pro)-Pro-Cys active site bearing two cysteine (Cys) residues that can ...be found either as free thiols (reduced TRXs) or linked together by a disulfide bond (oxidized TRXs) during the catalytic cycle. Their reactivity is crucial for TRX activity, and depends on the active site microenvironment. Here, we solved and compared the 3D structure of reduced and oxidized TRX h1 from
(CrTRXh1). The three-dimensional structure was also determined for mutants of each active site Cys. Structural alignments of CrTRXh1 with other structurally solved plant TRXs showed a common spatial fold, despite the low sequence identity. Structural analyses of CrTRXh1 revealed that the protein adopts an identical conformation independently from its redox state. Treatment with iodoacetamide (IAM), a Cys alkylating agent, resulted in a rapid and pH-dependent inactivation of CrTRXh1. Starting from fully reduced CrTRXh1, we determined the acid dissociation constant (p
) of each active site Cys by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analyses coupled to differential IAM-based alkylation. Based on the diversity of catalytic Cys deprotonation states, the mechanisms and structural features underlying disulfide redox activity are discussed.
•Many enzymes belonging to different chloroplast metabolic pathways are prone to redox control.•Thiol-based redox protein regulation includes mono-thiol and di-thiol modifications.•Nearly all the ...enzymes involved in starch degradation respond to redox modifications.•Primary starch is degraded at night under oxidizing conditions, while starch-degrading enzymes are mostly active under reducing conditions.•Redox control may have a minor role during starch degradation at night but could become significant in response to stress or in guard cells during stomatal opening.
Starch is one of the major sinks of fixed carbon in photosynthetic tissues of higher plants. Carbon fixation and the synthesis of primary starch occur during the day in the chloroplast stroma, whereas starch degradation typically occurs during the following night to fuel the whole plant with energy and carbon in the absence of photosynthesis. Redox-based regulatory systems play a central role in the modulation of several chloroplastic pathways. Reversible oxidations of cysteine residues are post-translational modifications that orchestrate the precise functioning of chloroplast pathways together with changes in pH, Mg2+ and concentrations of metabolic intermediates. Leaf starch metabolism has been intensively studied. The enzymes involved in starch synthesis and degradation have been identified and characterized. However, the redox control of the enzymes responsible for starch degradation at night remains elusive, and their response to redox transitions conflicts with the timing of the physiological events. Most of the enzymes of starch degradation are activated by reducing conditions, characteristic of daytime. Thus, redox control may have only a minor role during starch degradation at night, but could become relevant for daily stomatal opening in guard cells or in the re-allocation of fixed carbon in mesophyll cells in response to stress conditions.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two enzymes of the Calvin Benson cycle that stand out for some peculiar properties they have in common: (i) they ...both use the products of light reactions for catalysis (NADPH for GAPDH, ATP for PRK), (ii) they are both light-regulated through thioredoxins and (iii) they are both involved in the formation of regulatory supramolecular complexes in the dark or low photosynthetic conditions, with or without the regulatory protein CP12. In the complexes, enzymes are transiently inactivated but ready to recover full activity after complex dissociation. Fully active GAPDH and PRK are in large excess for the functioning of the Calvin-Benson cycle, but they can limit the cycle upon complex formation. Complex dissociation contributes to photosynthetic induction. CP12 also controls PRK concentration in model photosynthetic organisms like Arabidopsis thaliana and Chlamydomonas reinhardtii. The review combines in vivo and in vitro data into an integrated physiological view of the role of GAPDH and PRK dark complexes in the regulation of photosynthesis.
In many biogenic materials, chitin chains are assembled in fibrils that are wrapped by a protein fold. In them, the mechanical properties are supposed to be related to intra- and inter- interactions ...among chitin and proteins. This hypothesis has been poorly investigated. Here, this research theme is studied using the pen of Loligo vulgaris as a model material of chitin-protein composites. Chemical treatments were used to change the interactions involving only the proteic phase, through unfolding and/or degradation processes. Successively, structural and mechanical parameters were examined using spectroscopy, microscopy, X-ray diffractometry, and tensile tests. The data analysis showed that chemical treatments did not modify the structure of the chitin matrix. This allowed to derive from the mechanical test analysis the following conclusions: (i) the maximum stress (σmax) relies on the presence of the disulfide bonds; (ii) the Young's modulus (E) relies on the overall correct folding of the proteins; (iii) the whole removal of proteins induces a decrease of E (> 90%) and σmax (> 80%), and an increase in the maximum elongation. These observations indicate that in the chitin matrix the proteins act as a strengthener, which efficacy is controlled by the presence of disulfide bridges. This reinforcement links the chitin fibrils avoiding them to slide one on the other and maximizing their resistance and stiffness. In conclusion, this knowledge can explain the physio-chemical properties of other biogenic polymeric composites and inspire the design of new materials.
To date, no study has addressed on how proteins influence chitin-composite material's mechanical properties. Here we show that the Young's modulus and the maximum stress mainly rely on protein disulfide bonds, the inter-proteins ones and those controlling the folding of chitin-binding domains. The removal of protein matrix induce a reduction of Young's modulus and maximum stress, leaving the chitin matrix structurally unaltered. The measure of the maximum elongation shows that the chitin fibrils slide on each other only after removing the protein matrix.
In conclusion, this research shows that the proteins act as a stiff matrix reinforced by di-sulfide bridges that link crystalline chitin fibrils avoiding them to slide one on the other.
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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.
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