Encapsulins are self-assembling protein nanocompartments found in bacteria and archaea that encapsulate cargo enzymes to protect the cell from their toxic reaction products or intermediates. Developments in cryo-electron microscopy (cryo-EM) data processing strategies have enabled encapsulins and their cargo proteins to be investigated together in greater detail. In this study, we present the single particle cryo-EM structure of the Rhodospirillum rubrum encapsulin in both the presence and absence of its partner encapsulated ferritin (EncFtn). Single particle icosahedral reconstructions of empty and loaded encapsulins revealed a higher degree of conformational flexibility at the five-fold pore in the cargo loaded encapsulin. We applied a new non-point group averaging workflow to analyze the encapsulated ferritins within the encapsulin nanocompartment, to produce the first fully refined in situ atomic model of the EncFtn at 2.8 Å resolution. Masked 2D classification and particle subtraction demonstrate that cargo loading is heterogeneous in this recombinant complex, with the encapsulin able to house up to five of the decameric EncFtn complexes. Our data provides new insights into the dynamics and cargo arrangement in encapsulins and demonstrates an adaptable workflow for high resolution reconstruction of encapsulin cargoes.

Carbon-carbon bond formation is one of the key pillars of organic synthesis. Green, selective and efficient biocatalytic methods for such are therefore highly desirable. The α-oxoamine synthases (AOSs) are a class of pyridoxal 5'-phosphate (PLP)-dependent, irreversible, carbon-carbon bond-forming enzymes, which have been limited previously by their narrow substrate specificity and requirement of acyl-CoA thioester substrates. We recently characterized a thermophilic enzyme from Thermus thermophilus (ThAOS) with a much broader substrate scope and described its use in a chemo-biocatalytic cascade process to generate pyrroles in good yields and timescales. Herein, we report the structure-guided engineering of ThAOS to arrive at variants able to use a greatly expanded range of amino acid and simplified N-acetylcysteamine (SNAc) acyl-thioester substrates. The crystal structure of the improved ThAOS V79A variant with a bound PLP:L-penicillamine external aldimine ligand, provides insight into the properties of the engineered biocatalyst.

Chelators possess antibacterial properties linked to metal sequestration, simulating the action of nutritional immunity in preventing infection. To gain further insight into bacterial adaptation to metal restriction, we isolated mutants of Staphylococcus aureus with enhanced resistance to two synthetic chelators with therapeutic potential. Mutations were identified that altered peptidoglycan metabolism and teichoic acid modification, crucially affecting PBP2 and eliminating FmtA or VraF functionality. The resulting strains showed increased cell wall thickness, modified cell surface charge and varied in susceptibility to cell wall-targeting agents. In those mutants lacking either FmtA or VraF, the modifications substantially increased cell surface-associated calcium, offering protection against loss of manganese that was preferentially targeted by both chelators. Our phenotypic and cellular metal analyses identify the cell envelope of S. aureus as a key target for metal sequestering molecules. Peptidoglycan and teichoic acids, in particular, serve as key repositories for a subset of metal ions that safeguard against deprivation and can be altered to augment resistance to antibacterial chelators.

The aggregation of α-synuclein is a central neuropathological hallmark in neurodegenerative disorders known as Lewy body diseases, including Parkinson's disease and dementia with Lewy bodies. In the aggregation process, α-synuclein transitions from its native disordered/α-helical form to a β-sheet-rich structure, forming oligomers and protofibrils that accumulate into Lewy bodies, in a process that is thought to underlie neurodegeneration. Lipids are thought to play a critical role in this process by facilitating α-synuclein aggregation and contributing to cell toxicity, possibly through ceramide production. This study aimed to investigate biochemical changes associated with α-synuclein aggregation, focusing on lipid changes, using Raman spectroscopy coupled with machine learning. HEK293, Neuro2a and SH-SY5Y expressing increased levels of α-synuclein were treated with sonicated α-synuclein pre-formed fibrils, to model seeded aggregation. Raman spectroscopy, complemented by an in-house lipid spectral library, was used to monitor the aggregation process and its effects on cellular viability over 14 days. We detected α-synuclein aggregation by assessing β-sheet peaks at 1045 cm⁻1, in cells treated with α-synuclein pre-formed fibrils, using machine learning (principal component analysis and uniform manifold approximation and projection) analysis based on Raman spectral features. Changes in lipid profiles, and especially sphingolipids, including a decrease in sphingomyelin and increase in ceramides, were observed, consistent with oxidative stress and apoptosis. Altogether, our study informs on biochemical alterations that can be considered for the design of therapeutic strategies for Parkinson's disease and related synucleinopathies.

The toolbox of C-C bond forming biocatalysts is forever expanding as demand increases for new and sustainable routes to organic molecules. One class of enzymes, the pyridoxal 5'-phosphate (PLP)-dependent α-oxoamine synthases (AOSs), have recently attracted attention as an alternative biocatalyst to the already industrially-useful aldolases and carboligases. The AOSs are able to catalyse the irreversible formation of versatile aminoketone building blocks from readily-available, renewable feedstocks such as amino acids and carboxylic acids. A wild type, heat-stable ThAOS has been successfully coupled with the Knorr-pyrrole reaction (KPR) in a chemoenzymatic cascade to form 24 unique pyrroles. The synthetic utility was further expanded by a rational engineering campaign targeting a V79 residue which structural studies revealed was close to the active site. The V79 variants were more stable and displayed a much broader substrate scope, enabling the production of over 80 aminoketone targets. These included products derived from unnatural acids and inexpensive acyl-thioesters. This chapter aims to encourage the reader to implement the C-C bond forming ThAOS biocatalysts in their strategies for organic synthesis.

Laminarinase, an enzyme with a specific affinity for laminarin—a complex polysaccharide found in the cell walls of brown algae and select marine organisms—was investigated in this study. We cloned and characterized a gene encoding a putative glycoside hydrolase family 16 (GH16) laminarinase derived from the Jermuk hot spring metagenome. The resulting product, named Jermuk-LamM, represents a novel 1,3-β-d-glucanase with 48.1

Carboxysomes in cyanobacteria and certain proteobacteria enable efficient CO 2 fixation by encapsulating ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a semipermeable shell. Sequestered CA catalyze the rapid interconversion of CO 2 and HCO 3 − , supplying elevated levels of CO 2 to boost Rubisco carboxylation. Despite its essential role, the structure and encapsulation of CA within carboxysomes remain poorly understood. Here, we determined the molecular structure of α-carboxysomal CA from the model chemoautotrophic bacterium Halothiobacillus neapolitanus ( Hn CsoSCA). Hn CsoSCA adopts a trimer-of-dimers oligomeric structure without the incorporation of a zinc ion at its symmetric center. Using synthetic minishells, we demonstrate that Hn CsoSCA interacts with the CsoS1A shell hexamer and is incorporated into the minishells at the inner surface, independent of the CsoS2 linker protein. Hn CsoSCA truncations suggest nonspecific interactions between Hn CsoSCA and CsoS1A. We further show that Hn CsoSCA bridges Rubisco and the shell facets. Our study offers insights into the assembly and encapsulation mechanisms of α-carboxysomes and provides the framework for reprogramming carboxysome structures for synthetic biology and biotechnological applications.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a growing threat to global health, with recent efforts towards its eradication being reversed in the wake of the COVID-19 pandemic. Increasing resistance to gyrase-targeting second-line fluoroquinolone antibiotics indicates the necessity to develop both novel therapeutics and our understanding of M. tuberculosis growth during infection. ParDE toxin-antitoxin systems also target gyrase and are regulated in response to both host-associated and drug-induced stress during infection. Here, we present microbiological, biochemical, structural, and biophysical analyses exploring the ParDE1 and ParDE2 systems of M. tuberculosis H37Rv. The structures reveal conserved modes of toxin-antitoxin recognition, with complex-specific interactions. ParDE1 forms a novel heterohexameric ParDE complex, supported by antitoxin chains taking on two distinct folds. Curiously, ParDE1 exists in solution as a dynamic equilibrium between heterotetrameric and heterohexameric complexes. Conditional remodelling into higher order complexes can be thermally driven in vitro. Remodelling induces toxin release, tracked through concomitant inhibition and poisoning of gyrase activity. Our work aids our understanding of gyrase inhibition, allowing wider exploration of toxin-antitoxin systems as inspiration for potential therapeutic agents.

Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO2 fixation. The carboxysome uses a polyhedral protein shell made of hexamers, pentamers, and trimers to encapsulate Rubisco, increasing CO2 levels near Rubisco to enhance carboxylation. Despite their role in the global carbon cycle, the molecular mechanisms behind carboxysome shell assembly remain unclear. Here, we present a structural characterization of α-carboxysome shells generated from recombinant systems, which contain all shell proteins and the scaffolding protein CsoS2. Atomic-resolution cryo-electron microscopy of the shell assemblies, with a maximal size of 54 nm, unveil diverse assembly interfaces between shell proteins, detailed interactions of CsoS2 with shell proteins to drive shell assembly, and the formation of heterohexamers and heteropentamers by different shell protein paralogs, facilitating the assembly of larger empty shells. Our findings provide mechanistic insights into the construction principles of α-carboxysome shells and the role of CsoS2 in governing α-carboxysome assembly and functionality.

N‐carbamoyl‐β‐alanine amidohydrolase (CβAA) constitutes one of the most important groups of industrially relevant enzymes used in the production of optically pure amino acids and derivatives. In this study, a CβAA‐encoding gene from Rhizobium radiobacter strain MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrCβAA) showed a specific activity of 14 U·mg−1 using N‐carbamoyl‐β‐alanine as a substrate with an optimum activity at 55 °C and pH 8.0. In this work, we report also the first prokaryotic CβAA structure at a resolution of 2.0 Å. A discontinuous catalytic domain and a dimerisation domain attached through a flexible hinge region at the domain interface have been revealed. We identify key ligand binding residues, including a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291). Our results allowed us to explain the preference of the enzyme for linear carbamoyl substrates, as large and branched carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrCβAA from R. radiobacter MDC 8606 for the industrial production of L‐α‐, L‐β‐ and L‐γ‐amino acids. The structural analysis provides new insights on enzyme–substrate interaction, which shed light on engineering of CβAAs for high catalytic activity and broad substrate specificity.

We report a chemo-biocatalytic cascade for the synthesis of substituted pyrroles, driven by the action of an irreversible, thermostable, pyridoxal 5′-phosphate (PLP)-dependent, C–C bond-forming biocatalyst (ThAOS). The ThAOS catalyzes the Claisen-like condensation between various amino acids and acyl-CoA substrates to generate a range of α-aminoketones. These products are reacted with β-keto esters in an irreversible Knorr pyrrole reaction. The determination of the 1.6 Å resolution crystal structure of the PLP-bound form of ThAOS lays the foundation for future engineering and directed evolution. This report establishes the AOS family as useful and versatile C–C bond-forming biocatalysts.

The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus . We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the Vv RsbR: Vv RsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the Vv RsbR and Vv RsbS stoichiometry and a symmetry breach in the oxygen sensing domain of Vv RsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output – regulation of iron metabolism - for a stressosome complex.

The stressosome is a pseudo-icosahedral megadalton bacterial stress-sensing protein complex consisting of several copies of two STAS-domain proteins, RsbR and RsbS, and the kinase RsbT. Upon perception of environmental stress multiple copies of RsbT are released from the surface of the stressosome. Free RsbT activates downstream proteins to elicit a global cellular response, such as the activation of the general stress response in Gram-positive bacteria. The molecular events triggering RsbT release from the stressosome surface remain poorly understood. Here we present the map of Listeria innocua RsbR1/RsbS complex at resolutions of 3.45 Å for the STAS domain core in icosahedral symmetry and of 3.87 Å for the STAS domain and N-terminal sensors in D2 symmetry, respectively. The structure reveals a conformational change in the STAS domain linked to phosphorylation in RsbR. Docking studies indicate that allosteric RsbT binding to the conformationally flexible N-terminal sensor domain of RsbR affects the affinity of RsbS towards RsbT. Our results bring to focus the molecular events within the stressosome complex and further our understanding of this ubiquitous signaling hub.

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo-electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.