Biology subjects

Blikstad, C.

Publications and source records attributed to Blikstad, C..

11 recordsLinked to original sources

bioRxiv2026

Identifying Novel Targets of the Stringent Response in Plants and Cyanobacteria using chemoproteomics

Survival in dynamic environments requires photosynthetic organisms to rapidly sense and respond to stress. The stringent response, mediated by the signaling molecule guanosine-3,5-bisdiphosphate (ppGpp), is crucial for acclimation to environmental changes such as darkness and nitrogen limitation. While it has been extensively characterized in heterotrophic bacteria such as Escherichia coli, the molecular mechanisms and regulatory targets of ppGpp in photosynthetic organisms remain less understood. Here, we report large-scale chemoproteomic identification of ppGpp-binding proteins across plant chloroplasts and cyanobacteria, revealing both conserved and novel targets compared to E. coli. In plants, we found that ppGpp regulates pyrimidine metabolism by inhibiting the chloroplastic enzyme aspartate transcarbamoylase (PyrB). In cyanobacteria, we found that ppGpp activates glucose-1-phosphate adenylyltransferase (GlgC) involved in glycogen synthesis, activates citrate synthase (GltA), and induces carboxysome aggregation. These findings expand the known ppGpp regulatory network in photosynthetic organisms and provide a foundation for understanding how ppGpp coordinates adaptation to nutrient and environmental stresses.

systems biology↗

bioRxiv2026

Molecular mechanism of redox regulation of the alpha-carboxysomal carbonic anhydrase CsoSCA

Carboxysomes are protein-based organelles that form the core of the bacterial CO2-concentrating mechanism (CCM) by elevating CO2 levels around Rubisco. They encapsulate Rubisco and carbonic anhydrase (CA) within a protein shell that, after closure, excludes cytosolic reductants. Because cytosolic CA activity would short-circuit the CCM, CA activity must be confined to the carboxysome, yet how -carboxysomes achieve this has remained unknown. Here we show that CsoSCA, the -carboxysomal CA, is redox-regulated: inactive under reducing conditions, active under oxidizing. This regulation is mediated by a conserved vicinal cysteine pair distal from the active site. CryoEM structures of Halothiobacillus neapolitanus CsoSCA under active and inactive conditions, and of an inactive cysteine variant, reveal that redox conditions modulate global conformational dynamics that reorganize the active site for catalysis. These findings advance the understanding of -carboxysome regulation and couple CsoSCA activation to lumenal oxidation during carboxysome maturation.

biochemistry↗

bioRxiv2025

Atomic resolution structure of spinach rubisco reveals protons and dynamics

Photosynthetic organisms sustain life on Earth by storing solar energy in biomass. Central to this process is rubisco, the enzyme that catalyses the fixation of CO2 to ribulose-1,5-bisphosphate, providing the primary gateway for inorganic carbon into the biosphere. Rubiscos catalytic efficiency is a major determinant of crop productivity and global carbon flux, making it a longstanding target for protein engineering.1-4 Yet, attempts to enhance its performance through rational design have met limited success due to an incomplete understanding of rubiscos catalytic mechanism. Here, we report an atomic resolution (1.25 [A]) cryo-EM structure of spinach rubisco in complex with the transition-state analogue 2-carboxyarabinitol-1,5-bisphosphate. Supported by large-scale quantum/classical (QM/MM) calculations, our structural analysis reveals protonation equilibria within the active site and unexpected structural flexibility across large protein regions despite the exceptionally tight ligand binding. Our findings provide new insight into the complex interplay of protonation equilibria and conformational sampling, suggesting a novel basis for rubiscos rational redesign utilizing strategies that rely on a combination of dynamic and electrostatic control.

biochemistry↗

bioRxiv2025

Structural basis of ATP release and ion selectivity in Pannexin1 channels

This study investigated the structural basis of adenosine triphosphate (ATP) release and ion selectivity by Pannexin1 (Panx1). The microinjection of active SRC tyrosine kinase increased Panx1-mediated currents in Panx1-expressing oocytes. Furthermore, a mutation mimicking phosphorylation at Y308 turned Panx1 into a constitutively open ATP-releasing channel with broad ion permeability. Cryogenic electron microscopy showed that for most maps, the channel alternated between wide and narrow conformations. However, in the map of the phosphomimetic Y308E mutation, Panx1 remains in a wide ATP-releasing conformation. The pore-lining residues, W74 and R75, are flexible and create, in concert with the sequestered N-terminal helix, a path for ATP. The data point to phosphorylation as a reversible switch that activates ATP permeability by stabilizing Panx1 in the ATP-releasing conformation, which is crucial for developing modulators for diseases such as cancer and chronic pain.

biophysics↗

bioRxiv2024

Structure-guided engineering of α-ketoisocaproate dioxygenase increases isobutene production in Synechocystis sp. PCC 6803

Isobutene is a promising precursor for jet fuel due to its high energy density and favorable combustion properties. Light-driven bioproduction of isobutene has recently been investigated as an alternative strategy to crude oil refinement or fermentation-based manufacturing processes by harnessing the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the -ketoisocaproate dioxygenase (RnKICD) from Rattus norvegicus. However, the obtained production level was not sufficient, partially due the promiscuous activity of RnKICD. The enzyme catalyzes both the reaction with {rho}-hydroxyphenylpyruvate (HPP) for homogentisate formation, as well as the reaction with -ketoisocaproate (KIC), the precursor for isobutene synthesis. Here, to overcome this bottleneck step in the isobutene biosynthesis, protein engineering was employed to improve RnKICD activity and in vivo isobutene production. Purified RnKICD variants were characterized by measuring in vitro KIC and HPP consumption rates, as well as isobutene formation rate. The active site mutations F336V, N363A altered the KIC and HPP consumption rates, while the KIC-to-isobutene conversion ratio was only marginally affected. Besides, the RnKICD variants F336V, N363A and F336V/N363A exhibited a substantially enhanced substrate selectivity for KIC over HPP. Among the examined engineered Synechocystis strains, Syn-F336V showed a 4-fold improvement in isobutene production, compared to the base strain (Syn-RnKICD). Our findings reveal that residues F336 and N363 play a crucial role in substrate interactions, as targeted mutations at these sites shifted the substrate selectivity towards KIC and elevated the in vivo isobutene production levels significantly. We conclude that engineering the active site of RnKICD is a potent tool for improving isobutene bioproduction in Synechocystis. HighlightsO_LIPromiscuous -ketoisocaproate dioxygenase (KICD) from Rattus norvegicus was overproduced in Synechocystis sp. PCC 6803. C_LIO_LIProtein engineering was used to improve substrate selectivity. C_LIO_LIThe generated F336V variant led to a 4-fold increase in isobutene production in vivo. C_LI

bioengineering↗

bioRxiv2022

L-Aspartate oxidase provides new insights into fumarate reduction in anaerobic darkness in Synechocystis sp. PCC6803

Cyanobacteria are promising microbial hosts for production of various industrially relevant compounds, such as succinate, a central metabolite of the tricarboxylic acid cycle (TCA). Cyanobacteria have been engineered to produce succinate during photoautotrophic growth, and are also able to secrete it during anoxic fermentation conditions. It has been assumed that under anoxic darkness, succinate can be formed by reduction of fumarate catalyzed by the succinate dehydrogenase complex (SDH), however, no characterization of SDH regarding this activity has been performed. In this study, we address this issue by generating strains of the unicellular cyanobacterium Synechocystis PCC 6803 (Synechocystis) deficient in one or several subunits of SDH, and investigating succinate accumulation in these strains during dark anaerobic fermentation. The results showed higher succinate accumulation in SDH deletion strains than in the wild type, indicating a succinate dehydrogenase activity of SDH rather than fumarate reduction under these conditions. We further explored the possibility of another potential route for succinate formation from fumarate via L-aspartate oxidase (Laspo). The gene encoding Laspo in Synechocystis could not be inactivated, indicating an essential function for this enzyme. Using purified SynLaspo, we could demonstrate in vitro that in addition to L-aspartate oxidation the enzyme exhibits an L-aspartate-fumarate oxidoreductase activity. We therefore suggest that reduction of fumarate to succinate during anoxic darkness can be a byproduct of the Laspo reaction, which is the first step in biosynthesis of NAD cofactors. This work contributes to the understanding of cyanobacterial TCA cycle for future engineering and sustainable production of dicarboxylic acids.

biochemistry↗

bioRxiv2022

Rubisco forms a lattice inside alpha-carboxysomes

Bacteria employ microcompartments to sequester enzymatic processes, either for purposes of protecting cellular contents from reactive intermediates or as a way of increasing reaction efficiency. In these structures, a cargo of enzymes and accessory proteins is encased within a semi-permeable protein shell that permits passage of substrates and products but restricts movement of intermediates. In addition to their importance as a component of many bacterial species metabolisms, microcompartments have recently become a target of protein engineering. The shells can be reassembled from purified proteins, and the full operons can be functionally expressed outside their native prokaryotes and can remain functional following purification. Despite the importance of microcompartments in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of their architecture, ultrastructure, and spatial organization. Here, we employ cryo electron tomography to image -carboxysomes, a pseudo-icosahedral microcompartment responsible for carbon fixation. We have solved a high-resolution subtomogram average of the Rubisco cargo in situ, and determined a novel arrangement of the enzyme. We find that the H. neapolitanus Rubisco polymerizes in vivo, mediated by the small Rubisco subunit. These fibrils can further pack to form a lattice with six-fold pseudo-symmetry. This arrangement preserves freedom of motion and accessibility around the Rubisco active site and the binding sites for two other carboxysome proteins, CsoSCA (a carbonic anhydrase) and the disordered CsoS2, even at Rubisco concentrations exceeding 800 M. This characterization of Rubisco cargo inside the -carboxysome provides new insight into the balance between order and disorder in microcompartment organization.

biophysics↗

bioRxiv2021

Discovery of a carbonic anhydrase-Rubisco supercomplex within the alpha-carboxysome

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation - Rubisco and carbonic anhydrase - and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the -carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the -carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into -carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 [A] single-particle cryo-electron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCAs binding site overlaps with that of CsoS2 but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme, but also as a central hub for mediating assembly through protein interactions.

biochemistry↗

bioRxiv2020

Functional reconstitution of a bacterial CO2 concentrating mechanism in E. coli

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an engineered E. coli strain. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth depending on CCM components. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.One Sentence Summary A bacterial CO2 concentrating mechanism enables E. coli to fix CO2 from ambient air.Competing Interest StatementD.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. A.B.-E. is co-founder of b.fab. These companies were not involved in this research in any way. All other authors declare no competing interests.View Full Text

synthetic biology↗

bioRxiv2019

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins - structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

cell biology↗

bioRxiv2018

Genome-wide screening reveals a novel class of carbonic anhydrase-like inorganic carbon transporters in chemoautotrophic bacteria

Many bacterial autotrophs rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, including a 200+ MDa protein organelle called the carboxysome, a systematic screen of CCM components has not been carried out. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the {gamma}-proteobacterium H. neapolitanus. Our screen revealed an operon encoding a domain of unknown function (PFAM:PF10070) and putative cation transporter subunit (PFAM:PF00361) is critical for CCM function. These two proteins, which we name DabA and DabB for \"DABs accumulate bicarbonate,\" function as a heterodimeric, energy-coupled inorganic carbon pump in E. coli. Furthermore, DabA has distant homology to a {beta}-carbonic anhydrase and binds a zinc ion necessary for activity. Based on these results, we propose that DABs function as vectorial CAs coupled to cation gradients and serve as inorganic carbon pumps throughout prokaryotic phyla.

molecular biology↗