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The CRP-FNR (cAMP receptor protein-fumarate/nitrate reductase regulator) superfamily of transcriptional regulators includes the cyanobacterial master regulator NtcA, which orchestrates large responses of cyanobacteria to nitrogen scarcity. NtcA uses as allosteric activator 2-oxoglutarate (2OG), a signal of nitrogen poorness and carbon richness, and binds a co-activating protein (PipX) that shuttles between the signaling protein PII and NtcA depending on nitrogen richness, thus connecting PII signaling and gene expression regulation. Here, combining structural (X-ray crystallography of six types of crystals including NtcA complexes with DNA, 2OG, and PipX), modeling, and functional [electrophoretic mobility shift assays and bacterial two-hybrid (BACTH)] studies, we clarify the reasons for the exquisite specificity for the binding of NtcA to its target DNA, its mechanisms of activation by 2OG, and its co-activation by PipX. Our crystal structures of PipX-NtcA-DNA complexes prove that PipX does not interact with DNA, although it increases NtcA-DNA contacts, and that it stabilizes the active, DNA-binding-competent conformation of NtcA. Superimposition of this complex on a very recently reported cryo-electron microscopy structure of NtcA in a transcription activity complex with RNA polymerase (RNAP), shows that PipX binding helps recruit RNAP by PipX interaction with RNAP, particularly with its gamma and sigma (region 4) subunits, a structural prediction supported here by BACTH experiments., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recently, acyl plastoquinol (APQ) and plastoquinone-B (PQ-B), which are fatty acid esters of plastoquinol and plastoquinone-C respectively, have been identified as the major neutral lipids in cyanobacteria. In Synechocystis sp. PCC 6803, Slr2103 having homology with the eukaryotic enzyme for triacylglycerol (TAG) synthesis, diacylglycerol acyltransferase 2 (DGAT2), was identified as responsible for the synthesis of these plastoquinone-related lipids. On the other hand, TAG synthesis in cyanobacteria remains controversial due to the low accumulation level within cyanobacterial cells together with the high contamination level from the environment. In this study, to quantify more precisely and elucidate the relationship between the accumulation of neutral lipids and the presence or absence of DGAT2-like genes, plastoquinone-related lipids and TAG were analyzed directly from total lipids of six cyanobacterial species with different sets of genes encoding DGAT2-like proteins belonging to two distinct subclades. The results showed that the synthesis of these neutral lipids is highly dependent on clade A DGAT2-like proteins under the culture conditions used in this study, although accumulation level of TAG was quite low. In contrast to APQ highly abundant in saturated fatty acids, the fatty acid composition of TAG was species-specific and partly reflected the total lipid composition. Gloeobacter violaceus PCC 7421, which lacks a DGAT2-like gene, accumulated APQ with a high proportion of C18:0, suggesting APQ synthesis by an unidentified acyltransferase., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein ( holo -ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro . In cases where the KS-CLF can be accessed via E. coli heterologous expression, often the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli , which allowed us to study this minimal type II PKS in vitro . Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

Peroxidases belong to a group of enzymes that are widely found in animals, plants and microorganisms. These enzymes are effective biocatalysts for a wide range of oxidations on various substrates. This work presents a biochemical and structural characterization of a novel heme-containing peroxidase from Cyanobacterium sp. TDX16, CyanoPOX. This cyanobacterial enzyme was successfully overexpressed in Escherichia coli as a soluble, heme-containing monomeric enzyme. Although CyanoPOX shares relatively low sequence identity (37 %) with bovine lactoperoxidase, it displays comparable biochemical properties. CyanoPOX is most stable and active in slightly acidic conditions (pH 6-6.5) and moderately thermostable (melting temperature around 48 °C). Several compounds that are typical substrates for mammalian lactoperoxidases were tested to establish the catalytic potential of CyanoPOX. Potassium iodide showed the highest catalytic efficiency (126 mM -1  s -1 ), while various aromatic compounds were also readily converted. Structural elucidation of CyanoPOX confirmed the presence of a non-covalently bound b-type heme cofactor that is situated in the central core of the protein. Except for a highly similar overall structure, CyanoPOX also has a conserved active site pocket when compared with mammalian lactoperoxidases. Due to its catalytic properties and high expression in a bacterial host, this newly discovered peroxidase shows promise for applications., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Inspired by natural cryptic halogenation in C,C -bond formation, this study developed a synthetic approach combining biocatalytic bromination with transition-metal-catalyzed cross-coupling. Using the cyanobacterial Am VHPO, a robust and sustainable bromination-arylation cascade was created. Genetic modifications allowed enzyme immobilization, enhancing the compatibility between biocatalysis and chemocatalysis. This mild, efficient method for synthesizing biaryl compounds provides a foundation for future biochemo cascade reactions harnessing halogenation as a traceless directing tool.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of β(1-4) glycosidic bonds and have attracted considerable attention because of their potential for enhancing efficiency in degrading recalcitrant polymeric substrates, in synergism with hydrolytic enzymes. Fungal-derived LPMOs are the most prevalent type, while other taxonomic groups have been described as potential alternative sources of these enzymes. In the present study, we aimed to identify and characterize in silico a LPMO of cyanobacterial origin with putative functions in chitin depolymerization. A similarity search of sequences and conservation of domains with characterized LPMOs identified a 289 amino acid protein from the cyanobacterium Mastigocoleus testarum (Order Nostocales), likely belonging to the CAZy-AA10 class. This protein is referred to as MtLPMO10. Phylogenetic analysis revealed that MtLPMO10 is homologous to the protein Tma12 from the fern Tectaria macrodonta, with 52.11 % sequence identity, which was the first LPMO characterized as originating from the plant kingdom. The protein tertiary structure predicted by the AlphaFold server indicates structural features common to LPMOs, such as a histidine brace formed by His31 and His132 and an immunoglobulin-like domain composed of antiparallel beta strands. Molecular dynamics (MD) simulation allowed the assessment of the enzyme-substrate affinity, using an initial pose based on literature data. The MtLPMO10-chitin complex remained stable during 100ns of MD, while the MtLPMO10-cellulose complex dissociated within 30ns of MD. Additionally, there was a shorter Cu(I)-H4 distance in the protein-substrate complex compared to the Cu(I)-H1 distance (averages of 6.0 ± 0.7 Å and 7.9 ± 0.7 Å, respectively), suggesting a C4 regioselectivity. This study highlights the existence of lytic polysaccharide monooxygenases in cyanobacteria and paves the way for further investigations related to this enigmatic class of enzymes and their potential use in biotechnological applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

A circadian clock is reconstituted in vitro by incubating three proteins, KaiA, KaiB, and KaiC from the non-nitrogen-fixing cyanobacterium Synechococcus elongatus PCC 7942 in the presence of ATP. Leptolyngbya boryana is a filamentous cyanobacterium that grows diazotrophically under microoxic conditions. Among the aforementioned proteins, KaiC is the main clock oscillator belonging to the RecA ATPase superfamily. Genomic studies have revealed the presence of many genes encoding KaiC family ATPases in archaea and bacteria; however, very few have been analyzed in detail. For example, the L. boryana genome encodes two kaiC homologs designated as LbkaiC1 (LBWT_14830) and LbkaiC2 (LBWT_17950). LbKaiC1 is highly similar to KaiC from S. elongatus PCC 7942 compared with LbKaiC2. LbKaiC1 and LbKaiC2 were purified as Strep-tag fusion proteins. LbKaiC1 formed a hexamer and exhibited autophosphorylation, autodephosphorylation, and ATPase activities. Furthermore, it exhibited circadian phosphorylation rhythm in the presence of KaiA and KaiB from S. elongatus PCC 7942, indicating that LbKaiC1 is the central oscillator of the circadian clock in L. boryana. The temporal separation of nitrogen fixation from photosynthesis may be supported by the circadian rhythm generated by LbKaiC1 in L. boryana. LbKaiC2 had low ATPase activity, which depended on temperature, and its autophosphorylation activity was not detected like a circadian oscillator KaiC. Although the function of LbKaiC2 remains unknown, this work will provide comprehensive understanding of KaiC family ATPases., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cyanobacteria show promise as hosts for whole-cell biocatalysis. Their photoautotrophic metabolism can be leveraged for a sustainable production process. Despite advancements, performance still lags behind heterotrophic hosts. A key challenge is the limited ability to overexpress recombinant enzymes, which also hinders their biocatalytic efficiency. To address this, we generated large-scale expression libraries and developed a high-throughput method combining fluorescence-activated cell sorting (FACS) and deep sequencing in Synechocystis sp. PCC 6803 (Syn. 6803) to screen and optimize its genetic background. We apply this approach to enhance expression and biocatalyst performance for three enzymes: the ketoreductase LfSDR1M50, enoate reductase YqjM, and Baeyer-Villiger monooxygenase (BVMO) CHMO mut . Diverse genetic combinations yielded significant improvements: optimizing LfSDR1M50 expression showed a 17-fold increase to 39.2 U g cell dry weight (CDW) -1 . In vivo activity of Syn. YqjM was improved 16-fold to 58.7 U g CDW -1 and, for Syn. CHMO mut , a 1.5-fold increase to 7.3 U g CDW -1 was achieved by tailored genetic design. Thus, this strategy offers a pathway to optimize cyanobacteria as expression hosts, paving the way for broader applications in other cyanobacteria strains and larger libraries., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Organohalogen compounds exhibit wide-ranging bioactivities and potential applications. Understanding natural biosynthetic pathways and improving the production of halogenated compounds has garnered significant attention. Recently, the biosynthetic pathway of a cyanobacterial neurotoxin, aetokthonotoxin, was reported. It contains two unique enzymes: a single-component flavin-dependent halogenase AetF and a new type of nitril synthase AetD. The crystal structures of these enzymes in complex with their cofactors and substrates that were recently reported will be presented here. The AetF structures reveal a tri-domain architecture, the transfer direction of the hydride ion, a possible path to deliver the hypohalous acid, and the unusual bispecific substrate-recognition mode. The AetD structures demonstrate that the nitrile formation should occur through the action of a diiron cluster, implying that the enzyme should be capable of catalyzing the nitrile formation of alternative amino acids. This information is of central importance for understanding the mechanism of action as well as the applications of these two the-first-of-its-kind enzymes., (© 2024 Federation of European Biochemical Societies.)

Carotenoids are essential components of the photosynthetic apparatus and precursors of plant hormones, such as strigolactones (SLs). SLs are involved in various aspects of plant development and stress-response processes, including the establishment of root and shoot architecture. SL biosynthesis begins with the reversible isomerization of all- trans -carotene into 9- cis -β-carotene, catalysed by DWARF27 β-carotene isomerase (D27). Sequence comparisons have revealed the presence of D27-related proteins in photosynthetic eukaryotes and cyanobacteria lacking SLs. To gain insight into the evolution of SL biosynthesis, we characterized the activity of a cyanobacterial D27 protein ( Ca D27) from Cyanobacterim aponinum , using carotenoid-accumulating Escherichia coli cells and in vitro enzymatic assays. Our results demonstrate that Ca D27 is an all- trans / cis and cis / cis -β-carotene isomerase, with a cis / cis conversion preference. Ca D27 catalysed 13- cis /15- cis- , all- trans /9- cis -β-carotene, and neurosporene isomerization. Compared with plant enzymes, it exhibited a lower 9- cis -/all- trans -β-carotene conversion ratio. A comprehensive genome survey revealed the presence of D27 as a single-copy gene in the genomes of 20 out of 200 cyanobacteria species analysed. Phylogenetic and enzymatic analysis of Ca D27 indicated that cyanobacterial D27 genes form a single orthologous group, which is considered an ancestral type of those found in photosynthetic eukaryotes. This article is part of the theme issue 'The evolution of plant meta‌bolism'.

Current views of O 2 accumulation in Earth history depict three phases: The onset of O 2 production by ∼2.4 billion years ago; 2 billion years of stasis at ∼1 % of modern atmospheric levels; and a rising phase, starting about 500 million years ago, in which oxygen eventually reached modern values. Purely geochemical mechanisms have been proposed to account for this tripartite time course of Earth oxygenation. In particular the second phase, the long period of stasis between the advent of O 2 and the late rise to modern levels, has posed a puzzle. Proposed solutions involve Earth processes (geochemical, ecosystem, day length). Here we suggest that Earth oxygenation was not determined by geochemical processes. Rather it resulted from emergent biological innovations associated with photosynthesis and the activity of only three enzymes: 1) The oxygen evolving complex of cyanobacteria that makes O 2 ; 2) Nitrogenase, with its inhibition by O 2 causing two billion years of oxygen level stasis; 3) Cellulose synthase of land plants, which caused mass deposition and burial of carbon, thus removing an oxygen sink and therefore increasing atmospheric O 2 . These three enzymes are endogenously produced by, and contained within, cells that have the capacity for exponential growth. The catalytic properties of these three enzymes paved the path of Earth's atmospheric oxygenation, requiring no help from Earth other than the provision of water, CO 2 , salts, colonizable habitats, and sunlight., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The evolution of oxygenic photosynthesis during the Archean (4-2.5 Ga) required the presence of complementary reducing pathways to maintain the cellular redox balance. While the timing of the evolution of superoxide dismutases (SODs), enzymes that convert superoxide to hydrogen peroxide and O 2 , within bacteria and archaea is not resolved, the first SODs appearing in cyanobacteria contained copper and zinc in the reaction center (CuZnSOD). Here, we analyse growth characteristics, SOD gene expression (qRT-PCR) and cellular enzyme activity in the deep branching strain, Pseudanabaena sp. PCC7367, previously demonstrated to release significantly more O 2 under anoxic conditions. The observed significantly higher growth rates (p < 0.001) and protein and glycogen contents (p < 0.05) in anoxically cultured Pseudanabaena PCC7367 compared to control cultures grown under present-day oxygen-rich conditions prompted the following question: Is the growth of Pseudanabaena sp. PCC7367 correlated to atmospheric pO 2 and cellular SOD activity? Expression of sodB (encoding FeSOD) and sodC (encoding CuZnSOD) strongly correlated with medium O 2 levels (p < 0.001). Expression of sodA (encoding MnSOD) correlated significantly to SOD activity during the day (p = 0.019) when medium O 2 concentrations were the highest. The cellular SOD enzyme activity of anoxically grown cultures was significantly higher (p < 0.001) 2 h before the onset of the dark phase compared to O 2 -rich growth conditions. The expression of SOD encoding genes was significantly reduced (p < 0.05) under anoxic conditions in stirred cultures, as were medium O 2 levels (p ≤ 0.001), compared to oxic-grown cultures, whereas total cellular SOD activity remained comparable. Our data suggest that increasing pO 2 negatively impacts the viability of early cyanobacteria, possibly by increasing photorespiration. Additionally, the increased expression of superoxide-inactivating genes during the dark phase suggests the increased replacement rates of SODs under modern-day conditions compared to those on early Earth., (© 2024 The Author(s). Geobiology published by John Wiley & Sons Ltd.)

Based on current challenges of poor targeting and limited choices in chemical control methods of cyanobacterial blooms (CBs), identifying new targets is an urgent and formidable task in the quest for target-based algaecides. This study discovered N -acylamino saccharin derivatives exhibiting potent algicidal activity. Thus, using N -acylamino saccharin as the probes, glyceraldehyde-3-phosphate dehydrogenase from cyanobacterial ( Cy GAPDH) was identified as a new target of algaecides through the activity-based protein profiling (ABPP) strategy for the first time. Building upon the structure of Probe2 , a series of derivatives were designed and synthesized, with compound b6 demonstrating the most potent inhibitory activity against Cy GAPDH and Synechocystis sp. PCC6803 (IC 50 = 1.67 μM and EC 50 = 1.15 μM). Furthermore, the potential covalent binding model of b6 to the cysteine residue C154 was explored through covalent possibility prediction, LC-MS experiments, substrate competitive inhibition experiments, and molecular docking. Especially, the results revealed C154 as a crucial covalent binding site, with residues T184 and R11 forming robust hydrophobic interactions and H181 establishing significant hydrogen-bonding interactions with b6 , highlighting their potential as essential pharmacophores. In summary, this study not only identifies a novel target of algaecides for the control of CB but also lays the solid foundation for the development of targeted covalent algaecides.

Type III polyketide synthases (PKSs) catalyze the formation of a variety of polyketide natural products with remarkable structural diversity and biological activities. Despite significant progress in structural and mechanistic studies of type III PKSs in bacteria, fungi, and plants, research on type III PKSs in cyanobacteria is lacking. Here, we report structural and mechanistic insights into CylI, a type III PKS that catalyzes the formation of the alkylresorcinol intermediate in cylindrocyclophane biosynthesis. The crystal structure of apo-CylI reveals a distinct arrangement of structural elements that are proximal to the active site. We further solved the crystal structures of CylI in complexes with two substrate analogues at resolutions of 1.9 Å. The complex structures indicate that N259 is the key residue that determines the substrate preference of CylI. We also solved the crystal structure of CylI complexed with the alkylresorcinol product at a resolution of 2.0 Å. Structural analysis and mutagenesis experiments suggested that S170 functions as a key residue that determines cyclization specificity. On the basis of this result, a double mutant was engineered to completely switch the cyclization of CylI from aldol condensation to lactonization. This work elucidates the molecular basis of type III PKS in cyanobacteria and lays the foundation for engineering CylI-like enzymes to generate new products., (© 2024 The Protein Society.)

Aims: This study identifies a unique glutathione S-transferase (GST) in extremophiles using genome, phylogeny, bioinformatics, functional characterization, and RNA sequencing analysis., Methods and Results: Five putative GSTs (H0647, H0729, H1478, H3557, and H3594) were identified in Halothece sp. PCC7418. Phylogenetic analysis suggested that H0647, H1478, H0729, H3557, and H3594 are distinct GST classes. Of these, H0729 was classified as an iota-class GST, encoding a high molecular mass GST protein with remarkable features. The protein secondary structure of H0729 revealed the presence of a glutaredoxin (Grx) Cys-Pro-Tyr-Cys (C-P-Y-C) motif that overlaps with the N-terminal domain and harbors a topology similar to the thioredoxin (Trx) fold. Interestingly, recombinant H0729 exhibited a high catalytic efficiency for both glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB), with catalytic efficiencies that were 155- and 32-fold higher, respectively, compared to recombinant H3557. Lastly, the Halothece gene expression profiles suggested that antioxidant and phase II detoxification encoding genes are crucial in response to salt stress., Conclusion: Iota-class GST was identified in cyanobacteria. This GST exhibited a high catalytic efficiency toward xenobiotic substrates. Our findings shed light on a diversified evolution of GST in cyanobacteria and provide functional dynamics of the genes encoding the enzymatic antioxidant and detoxification systems under abiotic stresses., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Photosynthetic and chemosynthetic extremophiles have evolved adaptations to thrive in challenging environments by finely adjusting their metabolic pathways through evolutionary processes. A prime adaptation target to allow autotrophy in extreme conditions is the enzyme Rubisco, which plays a central role in the conversion of inorganic to organic carbon. Here, we present an extensive compilation of Rubisco kinetic traits from a wide range of species of bacteria, archaea, algae, and plants, sorted by phylogenetic group, Rubisco type, and extremophile type. Our results show that Rubisco kinetics for the few extremophile organisms reported up to date are placed at the margins of the enzyme's natural variability. Form ID Rubisco from thermoacidophile rhodophytes and form IB Rubisco from halophile terrestrial plants exhibit higher specificity and affinity for CO 2 than their non-extremophilic counterparts, as well as higher carboxylation efficiency, whereas form ID Rubisco from psychrophile organisms possess lower affinity for O 2 . Additionally, form IB Rubisco from thermophile cyanobacteria shows enhanced CO 2 specificity when compared to form IB non-extremophilic cyanobacteria. Overall, these findings highlight the unique characteristics of extremophile Rubisco enzymes and provide useful clues to guide next explorations aimed at finding more efficient Rubiscos., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Dimethylsulfoniopropionate (DMSP) is an abundant marine organosulfur compound with roles in stress protection, chemotaxis, nutrient and sulfur cycling and climate regulation. Here we report the discovery of a bifunctional DMSP biosynthesis enzyme, DsyGD, in the transamination pathway of the rhizobacterium Gynuella sunshinyii and some filamentous cyanobacteria not previously known to produce DMSP. DsyGD produces DMSP through its N-terminal DsyG methylthiohydroxybutyrate S-methyltransferase and C-terminal DsyD dimethylsulfoniohydroxybutyrate decarboxylase domains. Phylogenetically distinct DsyG-like proteins, termed DSYE, with methylthiohydroxybutyrate S-methyltransferase activity were found in diverse and environmentally abundant algae, comprising a mix of low, high and previously unknown DMSP producers. Algae containing DSYE, particularly bloom-forming Pelagophyceae species, were globally more abundant DMSP producers than those with previously described DMSP synthesis genes. This work greatly increases the number and diversity of predicted DMSP-producing organisms and highlights the importance of Pelagophyceae and other DSYE-containing algae in global DMSP production and sulfur cycling., (© 2024. The Author(s).)

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco-the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO 2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s -1 median versus <7 s -1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature., (© 2024. The Author(s).)

DesC1 and DesC2, which are fatty acid desaturases found in cyanobacteria, are responsible for introducing a double bond at the Δ9 position of fatty-acyl chains, which are subsequently esterified to the sn-1 and sn-2 positions of the glycerol moiety, respectively. However, since the discovery of these two desaturases in the Antarctic cyanobacterium Nostoc sp. SO-36, no further research has been reported. This study presents a comprehensive characterization of DesC1 and DesC2 through targeted mutagenesis and transformation using two cyanobacteria strains: Anabaena sp. PCC 7120, comprising both desaturases, and Synechocystis sp. PCC 6803, containing a single Δ9 desaturase (hereafter referred to as DesCs) sharing similarity with DesC1 in amino acid sequence. The results suggested that both DesC1 and DesC2 were essential in Anabaena sp. PCC 7120 and that DesC1, but not DesC2, complemented DesCs in Synechocystis sp. PCC 6803. In addition, DesC2 from Anabaena sp. PCC 7120 desaturated fatty acids esterified to the sn-2 position of the glycerol moiety in Synechocystis sp. PCC 6803., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Triceptides are a class of ribosomally synthesized and post-translationally modified peptides defined by an aromatic C(sp 2 ) to Cβ(sp 3 ) bond. The Gly-rich repeat family of triceptide maturases (TIGR04261) are paired with precursor peptides (TIGR04260) containing a Gly-rich core peptide. These maturases are prevalent in cyanobacteria and catalyze cyclophane formation on multiple Ω1-X2-X3 motifs (Ω1 = Trp and Phe) of the Gly-rich precursor peptide. The topology of the individual rings has not been completely elucidated, and the promiscuity of these enzymes is not known. In this study, we characterized all the cyclophane rings formed by the triceptide maturase OscB and show the ring topology is uniform with respect to the substitution at Trp-C7 and the atropisomerism (planar chirality). Additionally, the enzyme OscB demonstrated substrate promiscuity on Gly-rich precursors and can accommodate a diverse array of engineered sequences. These findings highlight the versatility and implications for using OscB as a biocatalyst for producing polycyclophane-containing peptides for biotechnological applications.

Cyanobacterial CO 2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO 2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO 2 concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO 2 fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO 2 . This creates a high CO 2 concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO 2 hydration. This method allows us to determine the kinetic parameters k cat , K M and k cat /K M for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO 2 and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well., (Copyright © 2024. Published by Elsevier Inc.)

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of proteins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for interrogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS). Here, a joint platform is built to probe such structural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor complex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers., Competing Interests: Competing interests No., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Until recently, the cyanobacterial phylum only included oxygenic photosynthesizer members. The discovery of Melainabacteria as a group of supposed non-photosynthetic cyanobacteria asked to revisit such scenario. From metagenomic data, we were able to identify sequences encoding putative ADP-glucose pyrophosphorylases (ADP-GlcPPase) from free-living and intestinal Melainabacteria. The respective genes were de novo synthesized and over-expressed in Escherichia coli. The purified recombinant proteins from both Melainabacteria species were active as ADP-GlcPPases, exhibiting V max values of 2.3 (free-living) and 7.1 U/mg (intestinal). The enzymes showed similar S 0.5 values (∼0.3 mM) for ATP, while the one from the intestinal source exhibited a 6-fold higher affinity toward glucose-1P. Both recombinant ADP-GlcPPases were sensitive to glucose-6P activation (A 0.5 ∼0.3 mM) and Pi and ADP inhibition (I 0.5 between 0.2 and 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. As far as we know, this is the first biochemical characterization of an active enzyme from Melainabacteria. This work contributes to a better understanding of the evolution of allosteric regulation in the ADP-GlcPPase family, which is critical for synthesizing the main reserve polysaccharide in prokaryotes (glycogen) and plants (starch). In addition, our results offer further information to discussions regarding the phylogenetic position of Melainabacteria., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Lakes Sagami and Tsukui are reservoirs constructed by connecting to the Sagami River. Because of eutrophication of the lakes, cyanobacteria have appeared every year. This review deals with phenomena related to occurrence of cyanobacteria that have been observed for 40 years since 1974 at the lakes. These 40 years of observations raised three interesting issues including the retention of cyanobacteria on their surfaces. These phenomena have been attributed to the usual factors, such as illuminance, nutrition and water temperature, but our research results suggested that they cannot be resolved without the introduction of another factor. We have attempted to elucidate various phenomena involving cyanobacteria in lake ecosystems by chemical ecological methods using volatile organic compounds (VOCs) produced by the cyanobacteria as indicators. One of the VOCs, β-cyclocitral, was significantly involved in the above phenomena, which was considered to be produced by the carotenoid cleavage dioxygenase (CCD) of the cyanobacteria. β-Cyclocitral was not produced in the two known CCDs, but two additional CCDs to Microcystis aeruginosa participated to produce the β-cyclocitral. These CCDs did not directly produce β-cyclocitral, but it was accumulated in cells as their precursors. The released β-cyclocitral underwent a Baeyer-Villiger-like oxidation. It was speculated that Microcystis activated the CCD genes through density stress and produced β-cyclocitral, which acted as an allelopathic substance. As a result, the number of cells of cyanobacteria decreased, and the resulting nitrogen and phosphorus were fed to the living cyanobacteria. It is postulated that this "quorum sensing" was functioning in the above-mentioned issues.

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and the spontaneously occurred V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified mRNAs likely regulated jointly by RNase E and an sRNA and potential 3' end-derived sRNAs. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Carbonic anhydrases (CAs, EC 4.2.1.1) have been studied for decades and have been classified as a superfamily of enzymes which includes, up to date, eight gene families or classes indicated with the Greek letters α, β, γ, δ, ζ, η, θ, ι. This versatile enzyme superfamily is involved in multiple physiological processes, catalysing a fundamental reaction for all living organisms, the reversible hydration of carbon dioxide to bicarbonate and a proton. Recently, the ι-CA (LCIP63) from the diatom Thalassiosira pseudonana and a bacterial ι-CA (BteCAι) identified in the genome of Burkholderia territorii were characterised. The recombinant BteCAι was observed to act as an excellent catalyst for the physiologic reaction. Very recently, the discovery of a novel ι-CAs (COG4337) in the eukaryotic microalga Bigelowiella natans and the cyanobacterium Anabaena sp. PCC7120 has brought to light an unexpected feature for this ancient superfamily: this ι-CAs was catalytically active without a metal ion cofactor, unlike the previous reported ι-CAs as well as all known CAs investigated so far. This review reports recent investigations on ι-CAs obtained in these last three years, highlighting their peculiar features, and hypothesising that possibly this new CA family shows catalytic activity without the need of metal ions.

Alkanes are ubiquitous in marine ecosystems and originate from diverse sources ranging from natural oil seeps to anthropogenic inputs and biogenic production by cyanobacteria. Enzymes that degrade cyanobacterial alkanes (typically C15-C17 compounds) such as the alkane monooxygenase (AlkB) are widespread, but it remains unclear whether or not AlkB variants exist that specialize in degradation of crude oil from natural or accidental spills, a much more complex mixture of long-chain hydrocarbons. In the present study, large-scale analysis of available metagenomic and genomic data from the Gulf of Mexico (GoM) oil spill revealed a novel, divergent AlkB clade recovered from genomes with no cultured representatives that was dramatically increased in abundance in crude-oil impacted ecosystems. In contrast, the AlkB clades associated with biotransformation of cyanobacterial alkanes belonged to 'canonical' or hydrocarbonoclastic clades, and based on metatranscriptomics data and compared to the novel clade, were much more weakly expressed during crude oil biodegradation in laboratory mesocosms. The absence of this divergent AlkB clade in metagenomes of uncontaminated samples from the global ocean survey but not from the GoM as well as its frequent horizontal gene transfer indicated a priming effect of the Gulf for crude oil biodegradation likely driven by natural oil seeps., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Evolution and function of glutathione S-transferase (GST) in primordial oxygenic phototrophs such as cyanobacteria are poorly understood. In this study, we identified and functionally characterized the GST gene family in the halotolerant cyanobacterium Halothece sp. PCC7418. Four putative Halothece-GSTs had very low homology, which implies evolutionary divergence. Of these, H0647, H0729 and H3557 were differentially expressed by oxidative stress whereas H3557 was highly and specifically upregulated under salt stress. In vitro analysis revealed that the recombinant H3557 exhibited GST activity toward 1-chloro-2, 4-dinitrobenzene (CDNB) and glutathione (GSH). H3557 displayed a broad range of activity at pH 6.5-10.5. Kinetic parameters showed the apparent K m for CDNB and GSH was 0.14 and 0.75 mM, respectively. H3557 remained catalytically active in the presence of NaCl. Structural modelling supported that H3557 is salt-adaptive enzyme with highly acidic residues on the protein surface. The vital function of H3557 in heterologous expression system was evaluated. The H3557-expressing cells were more tolerant to H 2 O 2 -induced oxidative stress compared with other GST-expressing cells and conferred salt tolerance. Taken together, the findings of this study provide insights into the molecular and cellular functions of GST in cyanobacteria, particularly under salt stress, which is less understood compared with other species., (© 2021 John Wiley & Sons Ltd.)

Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements 1 . However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion 2-7 . Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ 7 , but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Cyanobacteriochromes (CBCRs) are spectrally diverse photosensors from cyanobacteria distantly related to phytochromes that exploit photoisomerization of linear tetrapyrrole (bilin) chromophores to regulate associated signaling output domains. Unlike phytochromes, a single CBCR domain is sufficient for photoperception. CBCR domains that regulate the production or degradation of cyclic nucleotide second messengers are becoming increasingly well characterized. Cyclic di-guanosine monophosphate (c-di-GMP) is a widespread small-molecule regulator of bacterial motility, developmental transitions, and biofilm formation whose biosynthesis is regulated by CBCRs coupled to GGDEF (diguanylate cyclase) output domains. In this study, we compare the properties of diverse CBCR-GGDEF proteins with those of synthetic CBCR-GGDEF chimeras. Our investigation shows that natural diversity generates promising candidates for robust, broad spectrum optogenetic applications in live cells. Since light quality is constantly changing during plant development as upper leaves begin to shade lower leaves-affecting elongation growth, initiation of flowering, and responses to pathogens, these studies presage application of CBCR-GGDEF sensors to regulate orthogonal, c-di-GMP-regulated circuits in agronomically important plants for robust mitigation of such deleterious responses under natural growing conditions in the field., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring β -hydroxyaspartate ( β -OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of β -OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe 3+ to Fe 2+ photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia , named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread β -OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe 2+ for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth's ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, β -hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters.

The ancestors of cyanobacteria generated Earth's first biogenic molecular oxygen, but how they dealt with oxidative stress remains unconstrained. Here we investigate when superoxide dismutase enzymes (SODs) capable of removing superoxide free radicals evolved and estimate when Cyanobacteria originated. Our Bayesian molecular clocks, calibrated with microfossils, predict that stem Cyanobacteria arose 3300-3600 million years ago. Shortly afterwards, we find phylogenetic evidence that ancestral cyanobacteria used SODs with copper and zinc cofactors (CuZnSOD) during the Archaean. By the Paleoproterozoic, they became genetically capable of using iron, nickel, and manganese as cofactors (FeSOD, NiSOD, and MnSOD respectively). The evolution of NiSOD is particularly intriguing because it corresponds with cyanobacteria's invasion of the open ocean. Our analyses of metalloenzymes dealing with reactive oxygen species (ROS) now demonstrate that marine geochemical records alone may not predict patterns of metal usage by phototrophs from freshwater and terrestrial habitats., (© 2021. Crown.)

Thioredoxin reductases control the redox state of thioredoxins (Trxs)-ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Besides their catalysis, specific interactions between starch/glycogen processing enzymes and their substrates have been reported. Multiple branching enzyme (BE) isoforms, BE1, BE2, and BE3, have been found in a limited number of cyanobacterial species that are characterized by amylopectin accumulation. Seven surface binding sites (SBSs) located away from the active site have been identified in crystal structures of cyanobacterial BE1 from Crocosphaera subtropica (Cyanothece sp.) ATCC 51142 (51142BE1). In the present study, binding affinity toward amylopectin, amylose, and glycogen was investigated for wild-type 51142BE1 and its mutants (residues at SBSs important for sugar-binding were replaced by alanine). These enzymes showed retarded mobility during electrophoresis in non-denaturing polyacrylamide gels in the presence of polysaccharides. This was caused by interactions between the enzymes and the polysaccharides, enabling calculation of the dissociation constants (K d values) of the enzymes toward the polysaccharides. Mutational analysis indicated that particular domains of the protein (domains A and C) were involved in the polysaccharide binding. K d values toward the polysaccharides were also measured for 10 BE isoforms (five BE1, three BE2, and two BE3) from 5 cyanobacterial strains. All BEs displayed much lower K d values (higher affinity) toward amylopectin and amylose than toward glycogen, as described for plant BEs. In addition, one BE2 displayed exceptionally high K d values (low affinity), while two BE3 exhibited multiple K d values to all polysaccharides. These results could be ascribed to sequence variations in the SBSs, irrespective of the catalytic specificity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cyanobactins comprise a widespread group of peptide metabolites produced by cyanobacteria that are often diversified by post-translational prenylation. Several enzymes have been identified in cyanobactin biosynthetic pathways that carry out chemically diverse prenylation reactions, representing a resource for the discovery of post-translational alkylating agents. Here, genome mining was used to identify orphan cyanobactin prenyltransferases, leading to the isolation of tolypamide from the freshwater cyanobacterium Tolypothrix sp. The structure of tolypamide was confirmed by spectroscopic methods, degradation, and enzymatic total synthesis. Tolypamide is forward-prenylated on a threonine residue, representing an unprecedented post-translational modification. Biochemical characterization of the cognate enzyme TolF revealed a prenyltransferase with strict selectivity for forward O-prenylation of serine or threonine but with relaxed substrate selectivity for flanking peptide sequences. Since cyanobactin pathways often exhibit exceptionally broad substrate tolerance, these enzymes represent robust tools for synthetic biology., (© 2021 Wiley-VCH GmbH.)

Cyanobacteria are prolific producers of natural products, including polyketides and hybrid compounds thereof. Type III polyketide synthases (PKSs) are of particular interest, due to their wide substrate specificity and simple reaction mechanism, compared with both type I and type II PKSs. Surprisingly, only two type III PKS products, hierridins, and (7.7)paracyclophanes, have been isolated from cyanobacteria. Here, we report the mining of 517 cyanobacterial genomes for type III PKS biosynthesis gene clusters. Approximately 17% of the genomes analyzed encoded one or more type III PKSs. Together with already characterized type III PKSs, the phylogeny of this group of enzymes was investigated. Our analysis showed that type III PKSs in cyanobacteria evolved into three major lineages, including enzymes associated with 1) (7.7)paracyclophane-like biosynthesis gene clusters, 2) hierridin-like biosynthesis gene clusters, and 3) cytochrome b5 genes. The evolutionary history of these enzymes is complex, with some sequences partitioning primarily according to speciation and others putatively according to their reaction type. Protein modeling showed that cyanobacterial type III PKSs generally have a smaller active site cavity (mean = 109.035 Å3) compared with enzymes from other organisms. The size of the active site did not correlate well with substrate size, however, the "Gatekeeper" amino acid residues within the active site were strongly correlated to enzyme phylogeny. Our study provides unprecedented insight into the distribution, diversity, and molecular evolution of cyanobacterial type III PKSs, which could facilitate the discovery, characterization, and exploitation of novel enzymes, biochemical pathways, and specialized metabolites from this biosynthetically talented clade of microorganisms., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in chorismate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chorismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by Ab10 in meta position yielding 3-Cl-4-HBA, which is then transfered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Ab1 producing the monomeric ambigol building block 2,4-dichlorophenol.

Although RNA helicases are essentially ubiquitous and perform roles in all stages of RNA metabolism, phylogenetic analysis of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase family in a single phylum has not been performed. Here, we performed a phylogenetic analysis on DEAD-box helicases from all currently available cyanobacterial genomes, comprising a total of 362 helicase protein sequences from 280 strains. DEAD-box helicases belonging to three distinct clades were observed. Two clades, the CsdA (cold shock DEAD-box A)-like and RhlE (RNA helicase E)-like helicases, cluster with the homologous proteins from Escherichia coli . The third clade, the CrhR (cyanobacterial RNA helicase Redox)-like helicases, is unique to cyanobacteria and characterized by a conserved sequence motif in the C-terminal extension. Restricted distribution is observed across cyanobacterial diversity with respect to both helicase type and strain. CrhR-like and CsdA-like helicases essentially never occur together, while RhlE always occurs with either a CrhR-like or CsdA-like helicase. CrhR-like and RhlE-like proteins occurred in filamentous cyanobacteria of the orders Nostocales , Oscillatoriales and Synechococcales . Similarly, CsdA- and RhlE-like proteins are restricted to unicellular cyanobacteria of the genera Cyanobium and Synechococcus . In addition, the unexpected occurrence of RhlE in two Synechococcus strains suggests recent acquisition and evolutionary divergence. This study, therefore, raises physiological and evolutionary questions as to why DEAD-box RNA helicases encoded in cyanobacterial lineages display restricted distributions, suggesting niches that require either CrhR or CsdA RNA helicase activity but not both. Extensive conservation of gene synteny surrounding the previously described rimO-crhR operon is also observed, indicating a role in the maintenance of photosynthesis. The analysis provides insights into the evolution, origin and dissemination of sequences within a single gene family to yield divergent functional roles.

d-Amino acids can have major effects on the structure, proteolytic stability, and bioactivity of peptides. Proteusin radical S -adenosyl methionine epimerases regioselectively install such residues in ribosomal peptides to generate peptides with the largest number of d-residues currently known in biomolecules. To study their utility in synthetic biology, we investigated the substrate tolerance and substrate-product relationships of the cyanobacterial model epimerase OspD using libraries of point mutants as well as distinct extended peptides that were fused to an N-terminal leader sequence. OspD was found to exhibit exceptional substrate promiscuity in E. coli , accepting 15 different amino acids and converting peptides with a broad range of compositions, secondary structures, and polarities. Diverse single and multiple epimerization patterns were identified that were dictated by the peptide sequence. The data suggest major potential in creating genetically encoded products previously inaccessible by synthetic biology.

Background: The sesquiterpene germacrene A is a direct precursor of ß-elemene that is a major component of the Chinese medicinal herb Curcuma wenyujin with prominent antitumor activity. The microbial platform for germacrene A production was previously established in Saccharomyces cerevisiae using the germacrene A synthase (LTC2) of Lactuca sativa., Results: We evaluated the performance of LTC2 (LsGAS) as well as nine other identified or putative germacrene A synthases from different sources for the production of germacrene A. AvGAS, a synthase of Anabaena variabilis, was found to be the most efficient in germacrene A production in yeast. AvGAS expression alone in S. cerevisiae CEN.PK2-1D already resulted in a substantial production of germacrene A while LTC2 expression did not. Further metabolic engineering the yeast using known strategies including overexpression of tHMGR1 and repression of squalene synthesis pathway led to an 11-fold increase in germacrene A production. Site-directed mutagenesis of AvGAS revealed that while changes of several residues located within the active site cavity severely compromised germacrene A production, substitution of Phe23 located on the lateral surface with tryptophan or valine led to a 35.2% and 21.8% increase in germacrene A production, respectively. Finally, the highest production titer of germacrene A reached 309.8 mg/L in shake-flask batch culture., Conclusions: Our study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.

The emergence of resistance to frontline antibiotics has called for novel strategies to combat serious pathogenic infections. Methicillin-resistant Staphylococcus aureus [MRSA] is one such pathogen. As opposed to traditional antibiotics, bacteriostatic anti-virulent agents disarm MRSA, without exerting pressure, that cause resistance. Herein, we employed a thermophilic Thermotoga maritima tryptophan synthase ( Tm TrpB1) enzyme followed by an isonitrile synthase and Fe(II)-α-ketoglutarate-dependent oxygenase, in sequence as biocatalysts to produce antivirulent indole vinyl isonitriles. We report on conversion of simple derivatives of indoles to their C3-vinyl isonitriles, as the enzymes employed here demonstrated broader substrate tolerance. In toto , eight distinct L-Tryptophan derived α-amino acids (7) were converted to their bioactive vinyl isonitriles (3) by action of an isonitrile synthase (WelI1) and an Fe(II)-α-ketoglutarate-dependent oxygenase (WelI3) yielding structural variants possessing antivirulence against MRSA. These indole vinyl isonitriles at 10 μg/mL are effective as antivirulent compounds against MRSA, as evidenced through analysis of rabbit blood hemolysis assay. Based on a homology modelling exercise, of enzyme-substrate complexes, we deduced potential three dimensional alignments of active sites and glean mechanistic insights into the substrate tolerance of the Fe(II)-α-ketoglutarate-dependent oxygenase.

RuBisCO (Ribulose 1,5 bisphosphate carboxylase/oxygenase) by virtue of its dual specificity towards oxygen and carbon dioxide is an important rate-limiting step in photosynthesis and is believed to be the key factor for limited productivity of higher plants and algae. The photoautotrophic growth rate of cyanobacteria is a culmination of several factors including, rates of photosynthetic reactions, stress combating mechanisms and basic biomass generation metabolism in combination with optimal nutrient availability, irradiance, gaseous environment, etc. In case of cyanobacteria, the effect of RuBisCO in affecting the multiplication rate has been observed to show varied response. The current paper presents the RuBisCO activity of an early diverging cyanobacterium, Gloeobacter violaceus PCC 7421 and also compares the growth rates and RuBisCO activity of various cyanobacteria. A spectrophotometric estimation in a coupled enzyme assay system of the heterologous expressed G. violaceus PCC 7421 RuBisCO in E. coli, upon purification, revealed a carboxylation activity of LSu to be 5 nMol of phosphoglycerate min -1 mg -1 of protein, which is in coherence with the organism's slow growth rate. Further, the in vitro complementation of RbcL with RbcS in presence of RbcX of G. violaceus facilitated partial reconstitution of the protein and was hence found to cause a four-fold enhancement in its specific activity. The unique characteristics of the primitive cyanobacteria, such as, absence of thylakoids, lack of several photosystem constituting genes, slow carboxylation rate, pose limitation for its rapid multiplication. The RuBisCO carboxylation rate is observed as not the sole but an important parameter for obtaining optimal cell multiplication rates in photo-autotrophically multiplying cyanobacteria.

ATPases Associated with diverse cellular Activities (AAA+) are a superfamily of proteins that typically assemble into hexameric rings. These proteins contain AAA+ domains with two canonical motifs (Walker A and B) that bind and hydrolyze ATP, allowing them to perform a wide variety of different functions. For example, AAA+ proteins play a prominent role in cellular proteostasis by controlling biogenesis, folding, trafficking, and degradation of proteins present within the cell. Several central proteolytic systems (e.g., Clp, Deg, FtsH, Lon, 26S proteasome) use AAA+ domains or AAA+ proteins to unfold protein substrates (using energy from ATP hydrolysis) to make them accessible for degradation. This allows AAA+ protease systems to degrade aggregates and large proteins, as well as smaller proteins, and feed them as linearized molecules into a protease chamber. This review provides an up-to-date and a comparative overview of the essential Clp AAA+ protease systems in Cyanobacteria (e.g., Synechocystis spp), plastids of photosynthetic eukaryotes (e.g., Arabidopsis, Chlamydomonas), and apicoplasts in the nonphotosynthetic apicomplexan pathogen Plasmodium falciparum. Recent progress and breakthroughs in identifying Clp protease structures, substrates, substrate adaptors (e.g., NblA/B, ClpS, ClpF), and degrons are highlighted. We comment on the physiological importance of Clp activity, including plastid biogenesis, proteostasis, the chloroplast Protein Unfolding Response, and metabolism, across these diverse lineages. Outstanding questions as well as research opportunities and priorities to better understand the essential role of Clp systems in cellular proteostasis are discussed., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Fatty acid desaturases (FADs) represent a class of oxygen-dependent enzymes that dehydrogenate C-C bonds in the fatty acids (FAs) producing unsaturated CC double bonds that markedly change the properties of biological membranes. FADs are highly specific towards their acyl substrates, the position and configuration of the introduced double bonds. The double bond positioning of soluble acyl-carrier-protein Δ9-FADs was determined relative to the carboxyl end of a FA. Similar mode was suggested for the acyl-lipid Δ12-FADs (also known as ω6-FADs), however, their exact counting order remain unknown. Here we used monounsaturated odd- (17:1Δ 10 ) and even-chain (18:1Δ 11 ) FAs to show that acyl-lipid Δ12-FADs of, at least, two cyanobacterial species, Gloeobacter violaceus and Synechocystis sp. strain PCC 6803, use neither end of the fatty acid (Δ or ω) as a counting reference point; but count three carbons toward the methyl end from an existing double bond in the monoene precursors irrespective of a FA chain length., Competing Interests: Declaration of competing interest Authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The conversion of aldehydes to valuable alkanes via cyanobacterial aldehyde deformylating oxygenase is of great interest. The availability of fossil reserves that keep on decreasing due to human exploitation is worrying, and even more troubling is the combustion emission from the fuel, which contributes to the environmental crisis and health issues. Hence, it is crucial to use a renewable and eco-friendly alternative that yields compound with the closest features as conventional petroleum-based fuel, and that can be used in biofuels production. Cyanobacterial aldehyde deformylating oxygenase (ADO) is a metal-dependent enzyme with an α-helical structure that contains di‑iron at the active site. The substrate enters the active site of every ADO through a hydrophobic channel. This enzyme exhibits catalytic activity toward converting C n aldehyde to C n-1 alkane and formate as a co-product. These cyanobacterial enzymes are small and easy to manipulate. Currently, ADOs are broadly studied and engineered for improving their enzymatic activity and substrate specificity for better alkane production. This review provides a summary of recent progress in the study of the structure and function of ADO, structural-based engineering of the enzyme, and highlight its potential in producing biofuels., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)