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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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Mélanie
Loiodice
,
Elodie
Drula
,
Zak
Mciver
,
Svetlana
Antonyuk
,
Arnaud
Basle
,
Marcelo
Lima
,
Edwin A.
Yates
,
Dominic P.
Byrne
,
Jamie
Coughlan
,
Andrew
Leech
,
Shahram
Mesdaghi
,
Daniel J.
Rigden
,
Sophie
Drouillard
,
William
Helbert
,
Bernard
Henrissat
,
Nicolas
Terrapon
,
Gareth S. A.
Wright
,
Marie
Couturier
,
Alan
Cartmell
Diamond Proposal Number(s):
[18598, 30305, 21970, 32677, 28406]
Open Access
Abstract: 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.
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Feb 2025
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Cellular retinoic acid binding protein 2 (CRABP2) transports retinoic acid from the cytoplasm to the nucleus where it then transfers its cargo to retinoic acid receptor-containing complexes leading to activation of gene transcription. We demonstrate using purified proteins that CRABP2 is also a cyclin D3-specific binding protein and that the CRABP2 cyclin D3 binding site and the proposed CRABP2 nuclear localization sequence overlap. Both sequences are within the helix-loop-helix motif that forms a lid to the retinoic acid binding pocket. Mutations within this sequence that block both cyclin D3 and retinoic acid binding promote formation of a CRABP2 structure in which the retinoic acid binding pocket is occupied by an alternative lid conformation. Structural and functional analysis of CRABP2 and cyclin D3 mutants combined with AlphaFold models of the ternary CDK4/6-cyclin D3-CRABP2 complex supports the identification of an α-helical protein binding site on the cyclin D3 C-terminal cyclin box fold.
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Oct 2024
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Krios II-Titan Krios II at Diamond
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Thomas J.
Mccorvie
,
Douglas
Adamoski
,
Raquel A. C.
Machado
,
Jiazhi
Tang
,
Henry J.
Bailey
,
Douglas S. M.
Ferreira
,
Claire
Strain-Damerell
,
Arnaud
Basle
,
Andre L. B.
Ambrosio
,
Sandra M. G.
Dias
,
Wyatt W.
Yue
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.
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Apr 2024
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Krios III-Titan Krios III at Diamond
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Karla
Helena-Bueno
,
Mariia Yu.
Rybak
,
Chinenye L.
Ekemezie
,
Rudi
Sullivan
,
Charlotte R.
Brown
,
Charlotte
Dingwall
,
Arnaud
Basle
,
Claudia
Schneider
,
James P. R.
Connolly
,
James N.
Blaza
,
Bálint
Csörgő
,
Patrick J.
Moynihan
,
Matthieu G.
Gagnon
,
Chris H.
Hill
,
Sergey V.
Melnikov
Diamond Proposal Number(s):
[28576]
Open Access
Abstract: To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3,4,5,6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon–EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.
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Feb 2024
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I03-Macromolecular Crystallography
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Augustinas
Silale
,
Yiling
Zhu
,
Jerzy
Witwinowski
,
Robert E.
Smith
,
Kahlan E.
Newman
,
Satya P.
Bhamidimarri
,
Arnaud
Basle
,
Syma
Khalid
,
Christophe
Beloin
,
Simonetta
Gribaldo
,
Bert
Van Den Berg
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group have recently been shown to lack well-characterised OM attachment systems, but instead have OmpM, which could represent an ancestral tethering system in bacteria. Here, we have determined the structure of the most abundant OmpM protein from Veillonella parvula (diderm Firmicutes) by single particle cryogenic electron microscopy. We also characterised the channel properties of the transmembrane β-barrel of OmpM and investigated the structure and PG-binding properties of its periplasmic stalk region. Our results show that OM tethering and nutrient acquisition are genetically linked in V. parvula, and probably other diderm Terrabacteria. This dual function of OmpM may have played a role in the loss of the OM in ancestral bacteria and the emergence of monoderm bacterial lineages.
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Nov 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Ani
Paloyan
,
Armen
Sargsyan
,
Mariam D.
Karapetyan
,
Artur
Hambardzumyan
,
Sergei
Kocharov
,
Henry
Panosyan
,
Karine
Dyukova
,
Marina
Kinosyan
,
Anna
Kreuger
,
Cecilia
Piergentili
,
Will A.
Stanley
,
Karrera Y.
Djoko
,
Arnaud
Basle
,
Jon
Marles-Wright
,
Garabed
Antranikian
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: 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 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 dimerization 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.
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Aug 2023
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