Biology subjects

Libero, G.

Publications and source records attributed to Libero, G..

3 recordsLinked to original sources

bioRxiv2024

Inactivation of mitochondrial complex IV in Physcomitrium patens reveals the essential role of respiration in coordinating plants metabolism

Photosynthetic organisms use sunlight as energy source but rely on respiration during the night and in non-photosynthetic tissues. Respiration is also active in photosynthetically active cells, where its role is still unclear due to a lack of viable mutants. Plants lacking cytochrome c oxidase (complex IV) activity are generally lethal but were here isolated exploiting the possibility of generating knockout lines through vegetative propagation in the moss Physcomitrium patens. The mutants showed severely impaired growth, with an altered composition of the respiratory apparatus and increased electron transfer through the alternative oxidase. The light phase of photosynthesis remained largely unaffected while the efficiency of carbon fixation was moderately reduced. Transcriptomic and metabolomic analyses showed that the disruption of the cytochrome pathway had broad consequences for carbon and nitrogen metabolism. A major alteration in nitrogen assimilation was observed with a general reduction in amino acid abundance. A partial rescue of the growth could be obtained by growing the plants with an external supply of amino acids but not with sugars, demonstrating that respiration in plant photosynthetic cells plays an essential role at the interface between carbon and nitrogen metabolism and a key role in providing carbon skeletons for amino acid biosynthesis.

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↗