I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: 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 SusCDdex core and an associated glycoside hydrolase (GHdex) and surface glycan binding protein (SBGPdex). Via X-ray crystallography we solved high-resolution structures of SBGPdex in isolation and SusDdex and GHdex 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 SusCdex, SusDdex and GHdex, with substrate observed in both open and closed states of SusDdex. 3D variability analysis showed a minority of particles in the process of SusDdex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.
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Jul 2026
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Douglas S. M.
Ferreira
,
Katie
Mclennan
,
Calum
Diamond
,
Melanie
Vollmar
,
Wasim
Kiyani
,
D. Sean
Froese
,
Jola
Kopec
,
Henry J.
Bailey
,
Rod
Chalk
,
Arnaud
Basle
,
Jonathan M.
Elkins
,
Jesse
Coker
,
Wyatt W.
Yue
,
Thomas J.
Mccorvie
Diamond Proposal Number(s):
[34172]
Open Access
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.
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May 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948, 32736]
Open Access
Abstract: 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.
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May 2026
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948, 32736]
Open Access
Abstract: 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 centered 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.
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Mar 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: 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 have 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 re-shaped 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 analysed 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 optimised for one metal cofactor can evolve a new metal preference, under suitable selection pressure, through re-optimisation of its active site for catalytic reactivity of the new metal cofactor.
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Feb 2026
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: 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 GAPDH. 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.
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Dec 2025
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[28576]
Open Access
Abstract: Carboxysomes in cyanobacteria and certain proteobacteria enable efficient CO2 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 CO2 and HCO3−, supplying elevated levels of CO2 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 (HnCsoSCA). HnCsoSCA adopts a trimer-of-dimers oligomeric structure without the incorporation of a zinc ion at its symmetric center. Using synthetic minishells, we demonstrate that HnCsoSCA interacts with the CsoS1A shell hexamer and is incorporated into the minishells at the inner surface, independent of the CsoS2 linker protein. HnCsoSCA truncations suggest nonspecific interactions between HnCsoSCA and CsoS1A. We further show that HnCsoSCA 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.
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Nov 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Open Access
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.
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Jul 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Zak
Mciver
,
Alicia
Moraleda-Montoya
,
Zongjia
Chen
,
Ruwan
Epa
,
David
Starns
,
Matthew
Davy
,
Mikel
Garcia-Alija
,
Arnaud
Basle
,
Mario
Schubert
,
Didier
Ndeh
,
Beatriz
Trastoy
,
Spencer J.
Williams
,
Marcelo E.
Guerin
,
Alan
Cartmell
Diamond Proposal Number(s):
[18598, 30305, 21970]
Open Access
Abstract: 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 metabolise 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, with targets α1,2-2O-methyl L-fucoside linkages, a sugar residue a sugar 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 homologue, and reveal that the family operates with inverting stereochemistry. Using this 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.
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Jun 2025
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I04-Macromolecular Crystallography
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Open Access
Abstract: 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.
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Mar 2025
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