Biology subjects

Fermani, S.

Publications and source records attributed to Fermani, S..

7 recordsLinked to original sources

bioRxiv2022

Structural and functional characterization of chloroplast ribulose-5-phosphate-3-epimerase from the model green microalga Chlamydomonas reinhardtii

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 from Chlamydomonas reinhardtii (CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of an 8{beta}8 triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.

biochemistry↗

bioRxiv2022

Structural snapshots of nitrosoglutathione binding and reactivity underlying S-nitrosylation of photosynthetic GAPDH

S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols. GSNO can also induce protein S-glutathionylation but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes enzyme inhibition by specifically inducing S-nitrosylation. Treatment with reducing agents restores CrGAPA activity completely. While the cofactor NADP+ only partially protects from GSNO-mediated S-nitrosylation, the resultant inactivation is completely blocked by the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible of CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. Quantum mechanical/molecular mechanical calculations were performed to investigate the reaction mechanism of GSNO with CrGAPA Cys149 and to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.

plant biology↗

bioRxiv2021

Unravelling the regulation pathway of photosynthetic AB-GAPDH

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 of the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant formed by hetero-tetramers of A and B-subunits, and the homo-tetramer A4. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and for this reason is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of B-subunits. Reversible inactivation of AB-GAPDH occurs via oxidation of a cysteine pair located in the CTE, and substitution of NADP(H) with NAD(H) in the cofactor binding domain. These combined conditions lead to a change in the oligomerization state and enzyme inactivation. SEC-SAXS and single-particle cryoEM analysis disclosed the structural basis of this regulatory mechanism. Both approaches revealed that (A2B2)n-GAPDH oligomers with n=1, 2, 4 and 5 co-exist in a dynamic system. B-subunits mediate the contacts between adjacent A2B2 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, while the CTE of this subunit moves 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 in B-subunits active sites and hence inactive oligomer stabilization. Significance StatementIn land plants, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) unique sink of reducing power of the entire Calvin-Benson cycle, is finely regulated. Based on the redox state and substrates concentration, its heteromeric form AB-GAPDH oscillates between a fully active heterotetramer (A2B2) and inactive oligomers. Experimental evidence demonstrates that GAPDH inactivation depends on the formation of dimers, tetramers or pentamers of A2B2-modules, linked together by C-terminal extensions (CTE) of B-subunits that extrude from one modular tetramer and occupy two active sites of the adjacent one. This molecular mechanism along with the unexpected observed dynamism of the system, shed light on how the Calvin-Benson cycle is modulated in function of the light environmental changes.

biochemistry↗

bioRxiv2020

Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. The kinetic properties of CrGSNOR1 were determined and the high-resolution three-dimensional structures of the apo and NAD+-bound forms of the enzyme were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved 3D-folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed. One-sentence summaryGSNOR1 from Chlamydomonas reinhardtii displays an unusual variability of the catalytic zinc coordination environment and an unexpected resistance to thiol-based redox modifications

plant biology↗

bioRxiv2020

Acidic monosaccharides become incorporated into calcite single crystals

Carbohydrates, along with proteins and peptides, are known to represent a major class of biomacromolecules involved in calcium carbonate biomineralization. However, in spite of multiple physical or biochemical characterizations, the explicit role of saccharide macromolecules (long chains of carbohydrate molecules) is not yet understood in mineral deposition. In the present study we investigated the influence of two common acidic monosaccharides (MSs), which are the simplest form of carbohydrates and are represented here by glucuronic and galacturonic acids, on the formation of calcite crystals in vitro. We show that the size, morphology and microstructure of calcite crystals are altered when they are grown in the presence of these MSs. More importantly, MSs were found to become incorporated into the calcite crystalline lattice and induce anisotropic lattice distortions, a widely studied phenomenon in other biomolecules related to CaCO3 biomineralization but never before reported in the case of single MSs. Changes in the calcite lattice induced by MS incorporation were precisely determined by the technique of high-resolution synchrotron powder X-ray diffraction. We believe that the results of this research may deepen our understanding of the interaction of saccharide polymers with an inorganic host and shed light on the implications of carbohydrates for biomineralization processes.

bioengineering↗

bioRxiv2019

Glutathionylation primes soluble GAPDH for late collapse into insoluble aggregates

Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an alternative disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.

plant biology↗

bioRxiv2018

Photosynthetic CO2 assimilation: the last gap in the structural proteome is closed

In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central {beta}-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 [A] apart and connected by a flexible region exclusive to photosynthetic eukaryotes--the clamp loop--which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and increased number of random coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes. Significance StatementIn chloroplasts, five enzymes of the Calvin-Benson (CB) cycle are regulated by thioredoxins (TRXs). These enzymes have all been structurally characterized with the notable exception of phosphoribulokinase (PRK). Here, we determined the crystal structure of chloroplast PRK from two model photosynthetic organisms. Regulatory cysteines appear distant from each other and are linked by a long loop that is present only in plant-type PRKs and allows disulfide bond formation and subsequent conformational rearrangements. Structural comparisons with ancient PRKs indicate that the presence of flexible regions close to regulatory cysteines is a unique feature that is shared by TRX-dependent CB cycle enzymes, suggesting that the evolution of the PRK structure has resulted in a global increase in protein flexibility for photosynthetic eukaryotes.

plant biology↗