Myrciaria dubia (camu-camu) is an economically important Amazonian fruit species with severely limited genomic resources despite growing commercial interest and conservation needs. This study sequenced, assembled, and comprehensively analyzed the complete chloroplast (cp) genome of M. dubia and performed comparative genomic analyses across the order Myrtales, including codon usage, codon decoding mechanisms, repetitive elements, nucleotide diversity, and phylogenetic reconstruction. The cp genome spanned 158,664 bp with a typical quadripartite organization: a large single-copy region (LSC = 87,640 bp), a small single-copy region (SSC = 18,324 bp), and paired inverted repeats (IRA/IRB = 26,350 bp each). Functional annotation identified 135 genomic features, comprising 84 protein-coding genes, 37 tRNA genes, eight rRNA genes, and five pseudogenes. Codon usage analysis revealed a strong AT bias (62.50%), with 30 preferred codons (RSCU > 1.0) predominantly ending with A/T nucleotides. Codon decoding analysis assigned all 61 sense codons to five mechanistic categories, collectively achieving complete codon coverage across all 23 Myrtales accessions. Repetitive element analysis identified 69 simple sequence repeats in M. dubia (91.3% mononucleotide, predominantly adenine motifs in LSC intergenic spacers) and 439 long dispersed repeat pairs (97.5% palindromic, concentrated in the inverted repeat regions). Nucleotide diversity varied significantly across structural regions (SSC, mean π = 0.173; LSC, mean π = 0.084; IRA/IRB, mean π = 0.010–0.015), with the highest diversity window (π = 0.240) located in the SSC at approximately 117.7 kb. SWAN analysis of 78 protein-coding genes identified rpl22, ycf1, matK, ndhF, and ndhD as the most variable loci, with no gene exceeding ω = 1.0, indicating a pervasive purifying selection. Comparative analysis across 19 Myrtaceae accessions demonstrated complete gene collinearity and conserved IR boundary arrangement, with divergence concentrated in the SSC intergenic regions. Phylogenetic analysis positioned M. dubia as a sister species to Plinia trunciflora within the Myrteae tribe, nested in a well-supported monophyletic Myrtaceae clade (bootstrap = 100%). These findings provide the first complete cp genome resource for M. dubia and establish an essential foundation for conservation genetics, breeding programs, and evolutionary studies of this economically valuable Amazonian species.

ABSTRACT Polyethylene terephthalate (PET) waste represents a major environmental challenge due to limited recycling solutions. Thermophilic bacteria from geothermal environments harbor diverse enzymatic machinery adapted to extreme conditions, offering promising biocatalysts for plastic degradation; however, biological resources from Peru and other South American countries remain scarce. We characterized four bacterial strains isolated from two geothermal sites in Cajamarca, Peru, screened for PET hydrolysis at 50°C. Whole‐genome sequencing using hybrid assembly achieved near‐complete circular genomes. GTDB‐Tk classification identified three species: Neobacillus thermocopriae (strain 19A), Bacillus licheniformis (strains 16P and BI2), and Brevibacillus agri (strain BI8). Quantitative assays revealed that strain 16P achieved the highest mass loss (0.598%), followed by strain BI8 (0.449%). ATR‐FTIR analysis of the incubated sheets showed a significant reduction of the ester carbonyl index in strains 16P, 19A, and BI8 relative to both non‐incubated PET and an abiotic control, whereas strain BI2 did not differ from the controls, indicating preferential modification of ester bonds at the sheet surface. Genome mining and structure‐based homology searches identified multiple candidate enzymes similar to validated PETases and carboxylesterases, including PETase46‐like homologs in strains BI8 and 16P and a terephthalate‐active carboxylesterase homolog in strain 16P. Molecular docking supported the conservation of catalytic geometry and substrate‐binding sites in these candidates. This work represents one of the first systematic genomic and structural characterizations of putative PET‐hydrolases in Peruvian geothermal bacteria, expanding knowledge of extremophile diversity and advancing thermostable enzymes for sustainable plastic waste management.

Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (Ankistrodesmus sp.). Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg2+-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from Ankistrodesmus sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in Ankistrodesmus sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.

L-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-D-mannose 3',5'-epimerase (GME), L-galactose dehydrogenase (L-GalDH), and l-galactono-1,4-lactone dehydrogenase (L-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. MdL-GalDH shows that the way in which NAD(+) association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that C. intestinalis septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had CiSEPT2 occupying the terminal position were able to assemble into filaments via NC-interface association. The CiSEPT7-CiSEPT9 tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7 Å) provides a well-defined view of the central NC-interface. On the other hand, the CiSEPT7-CiSEPT9 G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that C. intestinalis may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.

Ribosome biogenesis (RB) is a highly conserved process across eukaryotes that results in the assembly of functional ribosomal subunits. Studies in Saccharomyces cerevisiae and Homo sapiens have identified numerous RB factors (RBFs), including the NIP7 protein, which is involved in late-stage pre-60S ribosomal maturation. NIP7 expression has also been observed in Chlamydomonas reinhardtii, highlighting its evolutionary significance. This study aimed to characterize the function of the NIP7 protein from C. reinhardtii (CrNip7) through protein complementation assays and a paromomycin resistance test, assessing its ability to complement the role of NIP7 in yeast. Protein interaction studies were conducted via yeast two-hybrid assay to identify potential protein partners of CrNip7. Additionally, rRNA modeling analysis was performed using the predicted structure of CrNip7 to investigate its interaction with rRNA. The study revealed that CrNip7 can complement the role of NIP7 in yeast, implicating CrNip7 in the biogenesis of the 60S ribosomal subunit. Furthermore, two possible partner proteins of CrNip7, UNC-p and G-patch, were identified through yeast two-hybrid assay. The potential of these proteins to interact with CrNip7 was explored through in silico analyses. Furthermore, nucleic acid interaction was also evaluated, indicating the involvement of the N- and C-terminal domains of CrNIP7 in interacting with rRNA. Collectively, our findings provide valuable insights into the RBFs CrNip7, offering novel information for comparative studies on RB among eukaryotic model organisms, shedding light on its evolutionary conservation and functional role across species.

Septins possess a conserved guanine nucleotide-binding (G) domain that participates in the stabilization of organized hetero-oligomeric complexes which assemble into filaments, rings and network-like structures. The fruit fly, Drosophila melanogaster, has five such septin genes encoding Sep1, Sep2, Sep4, Sep5 and Pnut. Here, we report the crystal structure of the heterodimer formed between the G-domains of Sep1 and Sep2, the first from an insect to be described to date. A G-interface stabilizes the dimer (in agreement with the expected arrangement for the Drosophila hexameric particle) and this bears significant resemblance to its human counterparts, even down to the level of individual amino acid interactions. On the other hand, a model for the G-interface formed between the two copies of Pnut which occupy the centre of the hexamer, shows important structural differences, including the loss of a highly favourable bifurcated salt-bridge network. Whereas wild-type Pnut purifies as a monomer, the reintroduction of the salt-bridge network results in stabilizing the dimeric interface in solution as shown by size exclusion chromatography and thermal stability measurements. Adaptive steered molecular dynamics reveals an unzipping mechanism for dimer dissociation which initiates at a point of electrostatic repulsion within the switch II region. Overall, the data contribute to a better understanding of the molecular interactions involved in septin assembly/disassembly.

Septins, often described as the fourth component of the cytoskeleton, are structural proteins found in a vast variety of living beings. They are related to small GTPases and thus, generally, present GTPase activity which may play an important (although incompletely understood) role in their organization and function. Septins polymerize into long non-polar filaments, in which each subunit interacts with two others by alternating interfaces, NC and G. In Saccharomyces cerevisiae four septins are organized in the following manner, [Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11]n in order to form filaments. Although septins were originally discovered in yeast and much is known regarding their biochemistry and function, only limited structural information about them is currently available. Here we present crystal structures of Cdc3/Cdc10 which provide the first view of the physiological interfaces formed by yeast septins. The G-interface has properties which place it in between that formed by SEPT2/SEPT6 and SEPT7/SEPT3 in human filaments. Switch I from Cdc10 contributes significantly to the interface, whereas in Cdc3 it is largely disorded. However, the significant negative charge density of the latter suggests it may have a unique role. At the NC-interface, we describe an elegant means by which the sidechain of a glutamine from helix α0 imitates a peptide group in order to retain hydrogen-bond continuity at the kink between helices α5 and α6 in the neighbouring subunit, thereby justifying the conservation of the helical distortion. Its absence from Cdc11, along with this structure's other unusual features are critically discussed by comparison with Cdc3 and Cdc10.

Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.

Abstract In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff–Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure–function relationships in the enzymes involved in vitamin C synthesis.

Although experimental protein-structure determination usually targets known proteins, chains of unknown sequence are often encountered. They can be purified from natural sources, appear as an unexpected fragment of a well characterized protein or appear as a contaminant. Regardless of the source of the problem, the unknown protein always requires characterization. Here, an automated pipeline is presented for the identification of protein sequences from cryo-EM reconstructions and crystallographic data. The method's application to characterize the crystal structure of an unknown protein purified from a snake venom is presented. It is also shown that the approach can be successfully applied to the identification of protein sequences and validation of sequence assignments in cryo-EM protein structures.

In order to fully understand any complex biochemical system from a mechanistic point of view, it is necessary to have access to the three-dimensional structures of the molecular components involved. Septins and their oligomers, filaments and higher-order complexes are no exception. Indeed, the spontaneous recruitment of different septin monomers to specific positions along a filament represents a fascinating example of subtle molecular recognition. Over the last few years, the amount of structural information available about these important cytoskeletal proteins has increased dramatically. This has allowed for a more detailed description of their individual domains and the different interfaces formed between them, which are the basis for stabilizing higher-order structures such as hexamers, octamers and fully formed filaments. The flexibility of these structures and the plasticity of the individual interfaces have also begun to be understood. Furthermore, recently, light has been shed on how filaments may bundle into higher-order structures by the formation of antiparallel coiled coils involving the C-terminal domains. Nevertheless, even with these advances, there is still some way to go before we fully understand how the structure and dynamics of septin assemblies are related to their physiological roles, including their interactions with biological membranes and other cytoskeletal components. In this review, we aim to bring together the various strands of structural evidence currently available into a more coherent picture. Although it would be an exaggeration to say that this is complete, recent progress seems to suggest that headway is being made in that direction.

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function.