Abstract
Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) act as oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here we ...show that the key determinant of Grx function is a distinct loop structure adjacent to the active site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its function from oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-type loop abolished FeS transferase activity and activated the oxidative half reaction of the oxidoreductase. The reductive half-reaction, requiring the interaction with a second GSH molecule, was enabled by switching additional residues in the active site. We explain how subtle structural differences, mostly depending on the structure of one particular loop, act in concert to determine Grx function.
Since the discovery of an autonomous iron-sulfur cluster (Fe-S) assembly machinery in mitochondria, significant efforts to examine the nature of this process have been made. The assembly of Fe-S ...clusters occurs in two distinct steps with the initial synthesis of 2Fe-2S clusters by a first machinery followed by a subsequent assembly into 4Fe-4S clusters by a second machinery. Despite this knowledge, we still have only a rudimentary understanding of how Fe-S clusters are transferred and distributed among their respective apoproteins. In particular, demand created by continuous protein turnover and the sacrificial destruction of clusters for synthesis of biotin and lipoic acid reveal possible bottlenecks in the supply chain of Fe-S clusters. Taking available information from other species into consideration, this review explores the mitochondrial assembly machinery of Arabidopsis and provides current knowledge about the respective transfer steps to apoproteins. Furthermore, this review highlights biotin synthase and lipoyl synthase, which both utilize Fe-S clusters as a sulfur source. After extraction of sulfur atoms from these clusters, the remains of the clusters probably fall apart, releasing sulfide as a highly toxic by-product. Immediate refixation through local cysteine biosynthesis is therefore an essential salvage pathway and emphasizes the physiological need for cysteine biosynthesis in plant mitochondria.
Microalgae represent a potential sustainable source of molecules and materials and the improvement of the knowledge of their metabolic regulation is essential to maximize their potential. Nutrients ...deprivation stimulates the accumulation of reserve lipids, triacylglycerols but also inhibits photosynthesis with a negative impact on biomass production and therefore overall productivity.
In this work, the seawater microalga Nannochloropsis gaditana was cultivated long term in a semi-continuous system where the concentrations of nitrogen or phosphorus were limiting but still sufficient to sustain growth indefinitely, to highlight the response to a long-term nutrient limitation and distinguishing it from the one to acute short-term stress. N. gaditana cells can acclimate to chronic nutrients limitation maintaining photosynthetic activity while also accumulating lipids. Both nitrogen and phosphorus limitation induced an increase of triacylglycerols content, although not by the induction of the synthesis of fatty acids but rather by modulating the fluxes of reduced carbon molecules toward lipid biosynthesis. Photosynthetic activity was maintained under P limitation while this was strongly affected by nitrogen depletion, where proteins of photosynthetic apparatus were largely reduced in content but still maintained their functionality and were able to achieve half of the biomass productivity with 30% of the nitrogen supply.
•N. gaditana cultures were kept under long term Nitrogen or phosphorus limitation.•Cell maintained biomass growth while also accumulating triacylglycerols.•Genes for fatty acids biosynthesis were not overexpressed.•Cells acclimated to nitrogen limitation to maintain biomass productivity.
SUMMARY
Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during ...development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild‐type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2‐hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre‐reduced roGFP2 in the reverse reaction.
Significance Statement
We identify Arabidopsis glutaredoxins GRXC3 and GRXC4 as type II membrane proteins in the secretory pathway and GRXC1 and GRXC2 as cytosolic enzymes with GRXC1 being attached to membranes through N‐terminal myristoylation. Cytosolic GRXC1 and GRXC2 and luminal GRXC3 and GRXC4 display distinct biochemical properties in their oxidoreductase activities that separate C1/C2 from C3/C4, which suggests that the proteins have adapted to their respective local redox environment and substrates
Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. ...Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe-S supply in grxs15 mutants.
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