Chitoporins (ChiPs) are chitooligosaccharide-specific outer membrane porins found in various Gram-negative bacteria and are critical for marine species that use chitin as a carbon and nitrogen source. Vibrio spp. ChiPs, exemplified by VhChiP from Vibrio harveyi, are trimeric OmpC-like 16-stranded β-barrels with a long pore-confining loop L3 that interacts exclusively with long chain chitooligosaccharides. Here, using crystallography and electrophysiology, we show that two atypical ChiPs, Escherichia coli ChiP (EcChiP) and Serratia marcescens ChiP (SmChiP), are monomeric 18-stranded β-barrels with shorter L3 loops, which display weaker and less selective substrate interactions compared with their Vibrio counterparts. Structure-based phylogenetic analysis of bacterial ChiPs resolves three clades: monomeric ChiPs; clade A, trimeric ChiPs with an N-plug; and clade B, trimeric ChiPs lacking the characterized helical N-plug. These architectural differences likely reflect environmental selection. Marine Vibrio spp., living in chitin-rich environments, possess trimeric ChiPs that enable rapid chitin uptake, whereas soil-borne S. marcescens and multihabitat E. coli encode monomeric ChiPs that we speculate are more suited to variable substrate availability.

Abstract The eukaryotic positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T, plays a central role in regulating RNA polymerase II (RNAPII). Phosphorylation of the RNAPII C-terminal domain (CTD) by P-TEFb promotes promoter proximal pause release and enables productive transcriptional elongation across many genes. Cyclin T mediates protein-protein interactions, several of which have been structurally characterised, that help to recruit and fine-tune P-TEFb activity to ensure a tight regulation of transcription. We have previously reported a set of halogenated chemical fragments termed FragLites that can prospectively identify protein interaction sites. Here, we report the FragLite map of cyclin T2, revealing binding sites corresponding to structurally defined cyclin T partners CDK9, AFF4, and HIV-1 Tat. Furthermore, we demonstrate the utility of FragLites in identifying a previously uncharacterised BRD4 binding site. By integrating FragLite clustering with biophysical analyses and AlphaFold3 modelling, we delineate the cyclin T-BRD4 interface. These analyses provide a comprehensive, chemically enriched fragment map highlighting functionally relevant sites to support future probe and modulator development to selectively target P-TEFb.

Abstract Mycobacteria synthesise the unusual glycan ᴅ-arabinan as a major component of the cell wall glycoconjugates arabinogalactan (AG) and lipoarabinomannan (LAM). We previously identified Dysgonomonas gadei , a member of the Bacteroidota, as capable of complete ᴅ-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo-α-ᴅ-arabinofuranosidase activity against AG and LAM, although their linkage specificities were unknown. Here, using defined synthetic substrates, we show that the three enzymes possess distinct linkage preferences. We also develop α-ᴅ-arabinofuranosyl cyclophellitol aziridines as covalent inhibitors and activity-based probes for GH172 enzymes. X-ray crystallography and cryo-EM to reveal strikingly different quaternary assemblies across the three homologues, while a 1.5 Å cryo-EM structure of dodecameric Dg67 covalently modified by an aziridine inhibitor identifies the catalytic nucleophile and provides direct structural support for a retaining mechanism. A BODIPY-tagged aziridine probe selectively labelled the three GH172 enzymes in D. gadei cell lysates. Together, these findings define functional and structural diversity within GH172 and establish chemical probes for profiling α-ᴅ-arabinofuranosidase activity in complex biological samples.

Bacteroides thetaiotaomicron ( B. theta ) is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilisation. Like other Bacteroides , B. theta has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated “lid” lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named “utilisomes”), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the B. theta dextran utilisome, with a SusCD dex core and an associated glycoside hydrolase (GH dex ) and surface glycan binding protein (SBGP dex ). Via X-ray crystallography we solved high-resolution structures of SBGP dex in isolation and SusD dex and GH dex bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualise open and closed states of the complex. Three occupied dextran binding sites were observed across SusC dex , SusD dex and GH dex , with substrate observed in both open and closed states of SusD dex . 3D variability analysis showed a minority of particles in the process of SusD dex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.

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.

Abstract Human methionine synthase (MTR) is an essential enzyme of one carbon metabolism. Consisting of a catalytic N-half and a cobalamin binding C-half, MTR utilises this intricate organometallic cofactor in the methyl transfer from methyltetrahydrofolate to homocysteine producing methionine. Cobalamin loading into MTR, and its subsequent activation, requires methylmalonic aciduria and homocystinuria Type D (MMADHC) protein and methionine synthase reductase (MTRR), respectively. However, the molecular basis of cobalamin binding and activation of human MTR aided by MMADHC and MTRR remains unknown. Here, using cryo-electron microscopy, we determine structures of human MTR in its apo, and cobalamin bound states. Apo MTR adopts a conformation where the two halves of the enzyme act independently with the C-half posed to bind cobalamin. Binding of cobalamin and its activation causes conformational changes in MTR that result in a flexible catalytically active state. AlphaFold predictions, validated by interaction studies, show that MMADHC interacts with the C-half of apo MTR to facilitate cobalamin loading. Unexpectedly we found that MTRR interacts at two distinct sites within the C-half of MTR which may aid in activation. Collectively these findings lay the groundwork to uncover the mechanisms through how MMADHC and MTRR coordinate cobalamin loading and activation of human MTR.

The acquisition of vitamin B12 and related cobamides is a key determinant for the fitness of Bacteroidota in the gut. Depending on the species, this uptake process relies on one to four transport systems centred on conserved core outer membrane (OM) complexes composed of the TonB-dependent transporter BtuB and the surface-exposed lipoprotein BtuG. Additionally, the surface-exposed lipoprotein BtuH, although not tightly associated with the BtuBG complex, contributes to cobamide uptake and provides a fitness advantage. Here, we report the functional and structural characterization of BtuJ1 from Bacteroides thetaiotaomicron (B. theta), an additional surface-exposed lipoprotein in B12 uptake loci. BtuJ1 binds vitamin B12 and cobinamide (an intermediate in B12 biosynthesis) with low nM affinity, conferring a fitness advantage in B12-limited environments. Regardless of B12 availability, BtuJ1 is the most abundant of the B12-transport components encoded by B. theta. Under B12-replete conditions, BtuJ1 binds the vitamin, generating a readily available pool for transfer to the core BtuBG transport systems during periods of B12 depletion as demonstrated by in vitro and in vivo B12 transfer experiments. Together, these findings expand the known functionalities of the diverse accessory OM proteins employed by Bacteroidota and underscore the sophisticated strategies these human gut commensals use to secure vitamin B12 in the competitive environment of the human gut.

Iron is an essential element that can be growth-limiting in microbial communities, particularly those present within host organisms. To acquire iron, many bacteria secrete siderophores, secondary metabolites that chelate ferric iron. These iron chelates can be transported back into the cell via TonB-dependent transporters in the outer membrane, followed by intracellular liberation of the iron. Pathogenic Escherichia coli and Salmonella produce siderophores during gut infection. In response to iron starvation, the human gut symbiont Bacteroides thetaiotaomicron upregulates an iron piracy system, XusABC, which steals iron-bound siderophores from the invading pathogens. Here, we investigated the molecular details of xenosiderophore uptake across the outer membrane by the XusAB complex. Our crystal and cryogenic electron microscopy structures explain how the XusB lipoprotein recognizes iron-bound xenosiderophores and passes them on to the XusA TonB-dependent transporter. Moreover, we show that Xus homologues can transport a variety of siderophores with different iron-chelating functional groups.

Changes in protein properties and functions are central to the evolution of life. Metalloproteins can evolve by changing their preference from one metal cofactor to another. Recently, we demonstrated that the widely distributed iron- or manganese-dependent superoxide dismutase (SodFM) family has undergone numerous metal-preference changes, including during evolutionary adaptation of pathogenic bacteria to altered metal availability within the host. Yet the underlying properties of metal-binding sites that control metalloenzyme metal preference are unclear, and thus, we lack an understanding of how enzymatic metal preference can be reshaped by evolution. Here, we used spectral features of bound iron or manganese, whose intensities reflect their oxidation state, to assess how their redox properties are tuned during SodFM evolution. We systematically analyzed the metal oxidation state across diverse SodFMs from multiple phylogenetic groups with different catalytic metal preferences, including those known to have undergone evolutionary metal-preference switching. We observed a striking relationship between resting oxidation state and catalytic metal preferences. Mutagenesis of second-sphere residues previously identified as determining metal preference revealed that they modulate metal-dependent activity and cofactor oxidation state in tandem, demonstrating these properties are linked. Together, these data argue that the differing SodFM metal preferences observed across the tree of life evolved through tuning of their redox properties by the secondary coordination sphere. This study gives insight into the process by which a metalloenzyme originally optimized for one metal cofactor can evolve a new metal preference, under suitable selection pressure, through re-optimization of its active site for catalytic reactivity of the new metal cofactor.

Acidic glycans are essential for the biology of multicellular eukaryotes. To utilize them, microbial life including symbionts and pathogens has evolved polysaccharide lyases (PL) that cleave their 1,4 glycosidic linkages via a β-elimination mechanism. PL family 33 (PL33) enzymes have the unusual ability to target a diverse range of glycosaminoglycans (GAGs), as well as the bacterial polymer, gellan gum. In order to gain more detailed insight into PL33 activities we recombinantly expressed 10 PL33 members derived from all major environments and further elucidated the detailed biochemical and biophysical properties of five, showing that their substrate specificity is conferred by variations in tunnel length and topography. The key amino acids involved in catalysis and substrate interactions were identified, and employing a combination of complementary biochemical, structural, and modeling approaches, we show that the tunnel topography is induced by substrate binding to the glycan. Structural and bioinformatic analyses revealed that these features are conserved across several lyase families as well as in mammalian GAG epimerases.

Bacteriophages are bacterial viruses that provide alternatives to small-molecule drugs to combat infections by antibiotic-resistant bacteria. To infect a bacterial host, a phage needs to bind to the bacterial surface via receptor binding proteins (RBPs), which are critical for determining host specificity. For functionally important receptors, the RBP–receptor interaction could be exploited via phage steering, where emerging bacterial resistance due to receptor modification could make bacteria less fit or virulent. Despite this, relatively little is known about RBP–receptor interactions. Here, we build on the recent discovery of coliphages that have the outer membrane (OM) lipopolysaccharide translocon LptDE as their terminal receptor and show via cryogenic electron microscopy that, surprisingly, the RBP of the small siphophage Oekolampad binds to a hitherto unobserved, open state of LptDE. The open lateral gate of LptD is occupied by a β-strand peptide originating from the degraded N-terminal jellyroll domain of LptD, suggesting the possibility of LptD inhibition via peptidomimetics. A structure of LptDE in complex with the superinfection exclusion (SE) protein Rtp45 of the Oekolampad-related phage Rtp shows a mechanism of SE where Rtp45-induced conformational changes in LptD resulting from steric clashes preclude RBP binding. Finally, analysis of spontaneous Oekolampad-resistant Escherichia coli mutants identifies mutations in LptD that abolish the LptDE–RBP interaction in vitro. SDS-EDTA sensitivity assays of the mutants show no major OM defects, consistent with largely preserved LptDE function, and suggesting that phage steering via LptDE might be challenging.

The Gram-negative β-barrel assembly machinery (BAM) complex catalyses the folding and membrane insertion of newly synthesized β-barrel outer membrane proteins. The BAM is structurally conserved, but most studies have focused on Gammaproteobacteria. Here, using single-particle cryogenic electron microscopy, quantitative proteomics and functional assays, we show that the BAM complex is distinct within the Bacteroidota. Cryogenic electron microscopy structures of BAM complexes from the human gut symbiont Bacteroides thetaiotaomicron (3.3 Å) and the human oral pathogen Porphyromonas gingivalis (3.2 Å) show similar, seven-component complexes of ~325 kDa. The complexes are mostly extracellular and comprise canonical BamA and BamD; an integral, essential outer membrane protein, BamG, that associates with BamA; and four surface-exposed lipoproteins: BamH-K. Absent from the BAM in Pseudomonadota, BamG-K form a large, extracellular dome that may confer additional functionality to enable the folding and assembly of β-barrel-surface-exposed lipoprotein complexes that are a hallmark of the Bacteroidota. Our findings develop our understanding of fundamental biological processes in an important bacterial phylum.

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.

Copper is an essential micronutrient for bacteria, needed for important copper enzymes such as terminal respiratory oxidases. However, in excess, copper is toxic to bacteria. This toxicity is caused by its ability to bind tightly to proteins through the formation of Cu-Cys and Cu-His bonds. To control toxicity, bacteria have evolved homeostatic systems to safely handle the copper they need while efficiently sequestering and effluxing excess copper ions. We previously found that GapA, the abundant glycolytic glyceraldehyde-3-phosphate dehydrogenase enzyme in the Staphylococcus aureus cytosol, becomes associated with copper within cells cultured in medium containing excess copper. We found that this association of GapA with copper resulted in inhibition of its enzyme activity. Here, we have characterised this binding of copper ions to S. aureus GapA in vitro to determine the mechanism of copper inhibition of GapA. We found that purified recombinant GapA binds a single Cu(I) ion with high affinity. Crystallographic structural determination showed association of this copper ion with two active site residues, Cys151 and His178, known to be important for catalysis. This observation was confirmed by characterisation of mutated variants lacking these residues, which showed reduced ability to bind Cu(I) ions. Finally, we demonstrated that the cytosolic copper metallochaperone, CopZ, exhibits a tighter affinity for Cu(I) and can remove copper from GapA in vitro. Together, our data demonstrate the mechanism by which excess copper binds to the S. aureus GapA enzyme and irreversibly inhibit its activity and how the cellular homeostasis system is capable of resolving this inhibition.

Rhamnogalacturonan II is one of the most complex plant cell wall carbohydrates and is composed of 13 different sugars and 21 different glycosidic linkages. It is abundant in fruit and indulgence foods, such as chocolate and wine, making it common in the human diet. The human colonic commensal Bacteroides thetaiotaomicron expresses a consortium of 22 enzymes to metabolize rhamnogalacturonan II, some of which exclusively target sugars unique to rhamnogalacturonan II. Several of these enzyme families remain poorly described, and, consequently, our knowledge of rhamnogalacturonan II metabolism is limited. Chief among the poorly understood activities is glycoside hydrolase (GH) family 139, which targets α1,2-2O-methyl L-fucoside linkages, a sugar residue not found in any other plant cell wall complex glycans. Although the founding enzyme BT0984 was placed in the RG-II degradative pathway, no GH139 structure or catalytic blueprint had been available. We report the crystal structures of BT0984 and a second homolog revealing that the family operates with inverting stereochemistry. Using these data, we undertook a mutagenic strategy, backed by molecular dynamics, to identify the important substrate binding and catalytic residues, mapping these residues throughout the GH139 family revealing the importance of the O2 methyl interaction of the substrate. We propose a catalytic mechanism that uses a non-canonical Asn as a catalytic base and shares similarity with L-fucosidases/L-galactosidases of family GH95.

Ribosome heterogeneity is a paradigm in biology, pertaining to the existence of structurally distinct populations of ribosomes within a single organism or cell. This concept suggests that structurally distinct pools of ribosomes have different functional properties and may be used to translate specific mRNAs. However, it is unknown to what extent structural heterogeneity reflects genuine functional specialization rather than stochastic variations in ribosome assembly. Here, we address this question by combining cryo-electron microscopy and tomography to observe individual structurally heterogeneous ribosomes in bacterial cells. We show that 70% of ribosomes in Psychrobacter urativorans contain a second copy of the ribosomal protein bS20 at a previously unknown binding site on the large ribosomal subunit. We then determine that this second bS20 copy appears to be functionally neutral. This demonstrates that ribosome heterogeneity does not necessarily lead to functional specialization, even when it involves significant variations such as the presence or absence of a ribosomal protein. Instead, we show that heterogeneous ribosomes can cooperate in general protein synthesis rather than specialize in translating discrete populations of mRNA.

The BAM (β-barrel assembly machinery) complex is an evolutionarily conserved, multiprotein machine that catalyses the folding and membrane insertion of newly synthesised β-barrel outer membrane (OM) proteins in Gram-negative bacteria. Based on Proteobacteria, bacterial BAM is also structurally conserved, with an essential BamAD core and up to three auxiliary periplasmic lipoproteins of poorly defined function. Here we show, using structural biology, quantitative proteomics and functional assays, that the BAM complex is radically different within the Bacteroidetes, a large and important phylum widely distributed within the environment and animal microbiomes. Cryogenic electron microscopy (cryo-EM) structures of BAM complexes from the human gut symbiont Bacteroides thetaiotaomicron and the human oral pathogen Porphyromonas gingivalis show similar, seven-component complexes of ~325 kDa in size with most of the mass in the extracellular space. In addition to canonical BamA and BamD, the complexes contain an integral OM protein named BamF that is essential and intimately associated with BamA, as well as four surface-exposed lipoproteins (SLPs) named BamG-J. Together, BamF-J form a large, extracellular dome that likely serves as an assembly cage for the β-barrel-SLP complexes that are a hallmark of the Bacteroidetes. Our data suggest that BAM functionality in Bacteroidetes is substantially expanded from that in Proteobacteria and underscores the importance of studying other phyla for a more complete understanding of fundamental biological processes. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Attaching and effacing pathogens overcome colonisation resistance by competing with metabolically similar organisms for limited resources. Enterohaemorrhagic E. coli (EHEC) utilises the pathogenicity island-encoded Accessory ʟ-arabinose Uptake (Aau) transporter to effectively colonise the mouse gut, hypothesised to be achieved via an enhanced capacity to scavenge ʟ-arabinose. Aau is regulated exclusively in response to ʟ-arabinose, but it is unclear how this system specifically benefits EHEC in vivo. Here, we show that Aau displays a > 200-fold higher affinity for the monosaccharide D-ribulose, over ʟ-arabinose. EHEC cannot grow on D-ribulose as a sole carbon source and this sugar does not trigger aau transcription. However, Aau effectively transports D-ribulose into the cell only in the presence of ʟ-arabinose, where it feeds into the pentose phosphate pathway, after phosphorylation by the ʟ-ribulokinase AraB, thus providing EHEC a significant fitness advantage. EHEC has therefore evolved a mechanism of hijacking the canonical ʟ-arabinose utilisation machinery to promote D-ribulose utilisation in vivo. Furthermore, Citrobacter rodentium encodes an analogous system that exclusively transports D-ribulose and metabolises it via a dedicated D-ribulokinase. These unique mechanisms of D-ribulose utilisation suggest that convergent evolution has driven the ability of distinct pathogenic species to exploit this nutrient during invasion of the gut niche.

Rhamnogalacturonan II is one of the most complex plant cell wall carbohydrates and is composed of 13 different sugars and 21 different glycosidic linkages. It is abundant in fruit and indulgence foods, such as chocolate and wine, making it common in the human diet. The human colonic commensal Bacteroides thetaiotaomicron expresses a consortium of 22 enzymes to metabolize rhamnogalacturonan II, some of which exclusively target sugars unique to rhamnogalacturonan II. Several of these enzyme families remain poorly described, and, consequently, our knowledge of rhamnogalacturonan II metabolism is limited. Chief among the poorly understood activities is glycoside hydrolase (GH) family 139, which targets α1,2-2O-methyl L-fucoside linkages, a sugar residue not found in any other plant cell wall complex glycans. Although the founding enzyme BT0984 was placed in the RG-II degradative pathway, no GH139 structure or catalytic blueprint had been available. We report the crystal structures of BT0984 and a second homolog revealing that the family operates with inverting stereochemistry. Using these data, we undertook a mutagenic strategy, backed by molecular dynamics, to identify the important substrate binding and catalytic residues, mapping these residues throughout the GH139 family revealing the importance of the O2 methyl interaction of the substrate. We propose a catalytic mechanism that uses a non-canonical Asn as a catalytic base and shares similarity with L-fucosidases/L-galactosidases of family GH95.

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.