I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Abstract: Sulfoquinovose (SQ) is a major biogenic sulfonated sugar whose degradation fuels microbial sulfur and carbon cycling. In the sulfoglycolytic Entner–Doudoroff (sulfo-ED) pathway, 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) is cleaved by KDSG aldolase to yield pyruvate and sulfolactaldehyde, yet the structure and mechanism of this enzyme have remained unclear. We report the biochemical and structural characterization of a metal-dependent KDSG aldolase from Pseudomonas putida using chemo-enzymatically synthesized KDSG. The enzyme forms a homohexamer, with a (β/α)8 TIM-barrel monomer assembling as a ‘dimer-of-trimers’. The enzyme exhibits optimal activity in the presence of Co2+ or Mn2+, consistent with other class II aldolases. Kinetic analysis revealed millimolar-range KM values for KDSG and modest cross-reactivity with the related glycolytic intermediate, 2-keto-3,6-deoxy-6-phosphogluconate (KDPG). Crystal structures of the apo and Co2+•pyruvate-bound forms (2.85 Å and 2.80 Å) show a metal-coordinated active site at the subunit interface, with conserved residues mediating metal binding and catalysis, providing insights into the mechanism of sulfonate-specific aldol cleavage. Sequence-similarity network and genome-context analyses show that KDSG aldolases are widespread among Proteobacteria and typically cluster with sulfo-ED pathway genes. These results define the structural and mechanistic basis of KDSG aldolases and inform on their roles in bacterial sulfur metabolism.
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Mar 2026
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Krios I-Titan Krios I at Diamond
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Merijn B. L.
Vriends
,
Elisha
Moran
,
Martín
Calvelo
,
Thomas
Hansen
,
Isabelle B.
Pickles
,
Xincheng
Xin
,
Marieke
Biezeno
,
Zachary W. B.
Armstrong
,
Maria J.
Ferraz
,
Lei
Li
,
Alice
Lilley
,
Ruth
Harvey
,
Dmitri V.
Filippov
,
Qinghua
Liao
,
Sybrin P.
Schröder
,
Gijsbert A.
Van Der Marel
,
Marta
Artola
,
Johannes M. F. G.
Aerts
,
James N.
Blaza
,
Jeroen D. C.
Codée
,
Carme
Rovira
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[28576]
Open Access
Abstract: Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by noncovalent binding and emerging resistance. We hypothesized that Oseltamivir aziridines would unite transition-state mimicry for tight binding, with aziridine-enabled covalent capture of the catalytic tyrosine, thereby supporting both therapy and activity-based quantification. Here, we present oseltamivir-based aziridines, inspired by cyclophellitol chemistry, that act as covalent inhibitors and activity-based probes via an N-acylaziridine warhead. Free-energy calculations, and NMR observations, indicate a 4H5 half-chair preference consistent with the NA transition state, and selected analogues inhibit multiple NA subtypes with low nanomolar binding constants. Diverse evidence establishes covalency: time-dependent inactivation, inhibitor washout, intact-mass shifts, MS/MS identification of a tyrosine adduct, and QM/MM reaction profiles, while cryoEM of N1 aligns with the proposed binding mode, revealing an elimination product. The inhibitors demonstrate formidable activity against diverse viral neuraminidases, including H5N1, and further enable imaging and quantification of active NA. With their dual therapeutic and diagnostic potential, these first-in-class inhibitors indeed benefit from transition state mimicry and covalency, and thus offer a powerful platform for antiviral development and neuraminidase imaging, addressing urgent global health needs in influenza treatment and prevention.
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Mar 2026
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I03-Macromolecular Crystallography
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Open Access
Abstract: Metalloprotein discovery is often made post hoc, in which activity studies following protein isolation reveal a metal-ion dependence. Herein we take a different approach to finding metalloproteins, by building on the discovery of copper-containing lytic polysaccharide monooxygenases (LPMOs), which include an N-terminal histidine as part of their sequence. This residue acts as a natural chelator for transition metal ions, irrespective of the structure of the protein. We report the method of signal strapping, where sequences of N-terminal signal peptides artificially appended with a histidine residue at their C-terminus are used to bootstrap a proteomic search. These searches return sequences of proteins with an N-terminal histidine capable of coordinating a metal ion. We exemplify the approach by the discovery and characterisation of four classes of bacterial metalloproteins, including two that we denote as anglerases reflecting their potential to capture transition metal ions from the bacterial environment.
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Oct 2025
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I04-Macromolecular Crystallography
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Thomas D.
Downes
,
S. Paul
Jones
,
James D.
Firth
,
John F.
Darby
,
Amelia K.
Gilio
,
Hanna F.
Klein
,
Xinyu
Wang
,
David C
Blakemore
,
Claudia
De Fusco
,
Stephen D.
Roughley
,
Lewis R.
Vidler
,
Maria A.
Whatton
,
Alison Jo-Anne
Woolford
,
Gail L.
Wrigley
,
Roderick E.
Hubbard
,
Gideon
Davies
,
Peter
O'Brien
,
Liang
Wu
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Fragment-based drug discovery is widely used in both academia and industry during the early stages of drug discovery. There is a growing interest in the design of 3-D fragments for inclusion in fragment libraries in order to increase chemical space coverage. We present herein the design and synthesis of 58 shape-diverse 3-D fragments that are prepared using just three modular synthetic methodologies. The 3-D fragments comprise a cyclic scaffold (cyclopentane, pyrrolidine, piperidine, tetrahydrofuran or tetrahydropyran) with one aromatic or heteroaromatic ring and possess properties within 'rule-of-three' fragment space. 3-D shape is assessed using principal moments of inertia analysis and conformational diversity is achieved by considering all conformations up to 1.5 kcal mol -1 above the energy of the global minimum energy conformer. Due to the modular nature of the fragment syntheses, these 3-D fragments are synthetically-enabled for fragment elaboration followon work, a key design feature. This modular, shape-diverse 3-D fragment collection has delivered privileged starting points across a spectrum of targets. Fragments from the set have been crystallographically validated in the SARS-CoV-2 main protease (M pro ) and the nonstructural protein 3 (Nsp3) (Mac1) as well as human glycosyltransferase MGATV, a major enzyme in the mammalian N-glycosylation pathway and a promoter of aggressive metastatic cancers, underscoring the breadth of biological space that can be explored.
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Sep 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Christian
Roth
,
Olga V.
Moroz
,
Suzan A. D.
Miranda
,
Lucas
Jahn
,
Elena V.
Blagova
,
Andrey A.
Lebedev
,
Dorotea R.
Segura
,
Mary A.
Stringer
,
Esben P.
Friis
,
Joao P. L.
Franco Cairo
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Endo-galactosaminidases are an underexplored family of enzymes involved in the degradation of galactosaminogalactan (GAG) and other galactosamine-containing cationic exopolysaccharides produced by fungi and bacteria. These exopolysaccharides are part of the cell wall and extracellular matrix of microbial communities. Currently, these galactosaminidases are found in three distinct CAZy families: GH114, GH135 and GH166. Despite the widespread occurrence of these enzymes in nearly all bacterial and fungal clades, only limited biochemical and structural data are available for these three groups. To expand our knowledge of endo-galactosaminidases, we selected several sequences predicted to encode endo-galactosaminidases and produced them recombinantly for structural and functional studies. Only very few predicted proteins could be produced in soluble form, and activity against bacterial Pel (pellicle) polysaccharide could only be confirmed for one enzyme. Here, we report the structures of two bacterial and one fungal enzyme. Whereas the fungal enzyme belongs to family GH114, the two bacterial enzymes do not lie in the current GH families but instead define a new family, GH191. During structure solution we realized that crystals of all three enzymes had various defects including twinning and partial disorder, which in the case of a more severe pathology in one of the structures required the design of a specialized refinement/model-building protocol. Comparison of the structures revealed several features that might be responsible for the described activity pattern and substrate specificity compared with other GAG-degrading enzymes.
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May 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Alexandra
Males
,
Olga V.
Moroz
,
Elena
Blagova
,
Astrid
Munch
,
Gustav H.
Hansen
,
Annette H.
Johansen
,
Lars H.
Østergaard
,
Dorotea R.
Segura
,
Alexander
Eddenden
,
Anne V.
Due
,
Martin
Gudmand
,
Jesper
Salomon
,
Sebastian R.
Sørensen
,
Joao Paulo L.
Franco Cairo
,
Mark
Nitz
,
Roland A.
Pache
,
Rebecca M.
Vejborg
,
Sandeep
Bhosale
,
David J.
Vocadlo
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-D-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering.
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Mar 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Mahima
Sharma
,
Nicholas
Pudlo
,
Michael A.
Järvå
,
Arashdeep
Kaur
,
Alan
John
,
Laura
Burchill
,
James P.
Lingford
,
Ruwan
Epa
,
Palika
Abayakoon
,
Nichollas E.
Scott
,
Johan P.
Turkenburg
,
Gideon J.
Davies
,
Eric C.
Martens
,
Ethan D.
Goddard-Borger
,
Spencer J.
Williams
Diamond Proposal Number(s):
[18598, 24948, 32736]
Open Access
Abstract: The production of short-chain fatty acids (SCFAs) by Firmicutes (Bacillota) within the human gastrointestinal tract is recognized as critical for gut health and the progression of a range of disease states. Firmicutes are the most diverse phylum of human gut bacteria and are highly studied, and are often specialized to degrade just a few polysaccharide substrates. Members of the Firmicutes include key bacteria that produce butyrate, an SCFA that is generally not produced by members of the other major phyla. Recently, it was shown that Eubacterium rectale, a widespread member of the Firmicutes belonging to the Clostridiales cluster XIVa, can grow on the unusual but ubiquitous plant-derived sugar SQ using a sulfoglycolytic sulfofructose transaldolase pathway. Here, we show that in addition to SQ, E. rectale can also grow on the SQ glycoside sulfoquinovosyl glycerol (SQGro). The 3D structure of the E. rectale sulfoquinovosidase (SftG) shares strong structural conservation with other carbohydrate active enzyme family GH31 SQases. Using sequence-similarity networks, we provide new biological context to a conserved domain of unknown function protein SftX belonging to DUF4867, which is conserved in the sulfoglycolytic sulfofructose transaldolase pathway, and determine its 3D structure. Finally, with the aid of a synthetic mini-human microbiome reconstituted in germ-free mice, we show that an SQ dietary supplement can rescue E. rectale from population crashes that occur upon switching from a high-fiber to a low-fiber, high-fat diet. This suggests that SQ or SQGro has potential as a prebiotic for promoting the maintenance of this important butyrate-producing bacterium within the colonic microbiota.
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Feb 2025
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I03-Macromolecular Crystallography
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Gijs
Ruijgrok
,
Wendy A.
Offen
,
Isabelle B.
Pickles
,
Deepa
Raju
,
Thanasis
Patsos
,
Casper
De Boer
,
Tim
Ofman
,
Joep
Rompa
,
Daan
Van Oord
,
Eleanor J.
Dodson
,
Alexander
Beekers
,
Thijs
Voskuilen
,
Michela
Ferrari
,
Liang
Wu
,
Antonius P. A.
Janssen
,
Jeroen D. C.
Codée
,
P. Lynne
Howell
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[32736]
Open Access
Abstract: During infection, the human opportunistic pathogen Pseudomonas aeruginosa forms protective biofilms, whose matrix consists of proteins, nucleic acids, and polysaccharides such as alginate, Psl, and Pel. Psl, a polymeric pentasaccharide composed of mannose, rhamnose, and glucose, is produced during the early stages of biofilm formation, serving as a protective barrier against antibiotics and the immune system. The Psl biosynthesis gene cluster, besides encoding various glycosyltransferases, also includes an endoglycosidase, PslG. Here, we show, by activity-based protein profiling, structural studies on enzyme–inhibitor complexes, and defined substrate processing, that PslG is not, as previously suggested, an endo-β-mannosidase but instead a retaining endo-β-glucosidase. This insight allows the design of both competitive and covalent PslG inhibitors, as we show for repeating pentasaccharide mimetics featuring either a reducing end deoxynojirimycin or cyclophellitol moiety. This work provides valuable tools to deepen the understanding of Psl biosynthesis, its function in biofilm formation, and its contribution to antibiotic resistance. We demonstrate the enzyme’s actual endo−β–glucosidase activity, a means to monitor PslG activity in P. aeruginosa biofilms, and a blueprint for inhibitor design.
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Feb 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Open Access
Abstract: The sulfosugar sulfoquinovose (SQ) is catabolized through the sulfoglycolytic Entner-Doudoroff pathway, beginning with the oxidation of SQ to sulfogluconolactone by SQ dehydrogenase. We present a comprehensive structural and kinetic characterization of Pseudomonas putida SQ dehydrogenase (PpSQDH). PpSQDH is a tetrameric enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily with a strong preference for NAD+ over NADP+. Kinetic analysis revealed a rapid equilibrium ordered mechanism in which the NAD+ cofactor is the first substrate to bind, and NADH is the last product to dissociate. Structural studies revealed a homotetrameric structure in solution and crystals, involving cross-subunit interactions in which the C-terminus residue (Gln260) inserts into the diagonally opposite subunit to form part of the second shell of residues lining the active site. Complexes of PpSQDH with SQ or NAD+ provide insight into the recognition of SQ and together with the kinetic analysis allow the proposal of a catalytic reaction mechanism. Our findings illuminate the mechanism of SQ degradation and the evolution of the SDR superfamily for organosulfonate catabolism.
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Jan 2025
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I03-Macromolecular Crystallography
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Isabelle B.
Pickles
,
Yurong
Chen
,
Olga
Moroz
,
Haley A.
Brown
,
Casper
De Boer
,
Zachary
Armstrong
,
Nicholas G. S.
Mcgregor
,
Marta
Artola
,
Jeroen D. C.
Codée
,
Nicole M.
Koropatkin
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[24948, 32736]
Open Access
Abstract: α-Amylases are the workhorse enzymes of starch degradation. They are central to human health, including as targets for anti-diabetic compounds, but are also the key enzymes in the industrial processing of starch for biofuels, corn syrups, brewing and detergents. Dissection of the activity, specificity and stability of α-amylases is crucial to understanding their biology and allowing their exploitation. Yet, functional characterization lags behind DNA sequencing and genomics; and new tools are required for rapid analysis of α-amylase function. Here, we design, synthesize and apply new branched α-amylase activity-based probes. Using both α-1,6 branched and unbranched α-1,4 maltobiose activity-based probes we were able to explore the stability and substrate specificity of both a panel of human gut microbial α-amylases and a panel of industrially relevant α-amylases. We also demonstrate how we can detect and annotate the substrate specificity of α-amylases in the complex cell lysate of both a prominent gut microbe and a diverse compost sample by in-gel fluorescence and mass spectrometry. A toolbox of starch-active activity-based probes will enable rapid functional dissection of α-amylases. We envisage activity-based probes contributing to better selection and engineering of enzymes for industrial application as well as fundamental analysis of enzymes in human health.
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Nov 2024
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