I03-Macromolecular Crystallography
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Rob F.
Lammers
,
Qin
Su
,
Laura
Mazo
,
Wendy A.
Offen
,
Roelof
Ottenhoff
,
Mats J.
Bulterman
,
Martijn
Van Der Lienden
,
Maria J.
Ferraz
,
Florian
Küllmer
,
Zahid
Ilhan
,
Hans
Van Den Elst
,
Ingeborg
Van Der Made
,
Cindy P. A. A.
Van Roomen
,
Anne
De Jongh
,
Adrianus M. C. H.
Van Den Nieuwendijk
,
Rolf G.
Boot
,
Noam
Zelcer
,
Gideon J.
Davies
,
Carme
Rovira
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
,
Marta
Artola
Diamond Proposal Number(s):
[39189]
Open Access
Abstract: Neutral glucosylceramidase GBA2 is a retaining β-d-glucosidase involved in sphingolipid metabolism and implicated in neurological lysosomal storage disorders, yet the lack of highly selective chemical inhibitors has hindered efforts to disentangle its function from that of glucosylceramide synthase (GCS) and lysosomal glucosylceramidase (GBA1). Here, we report the development of the first brain-penetrant, covalent inhibitor that selectively inactivates GBA2 in vivo. Building on β-d-arabinofuranose-configured (β-d-Araf) cyclitol aziridine activity-based probes previously shown to covalently and selectively label GBA2, we designed and synthesized a focused library of electrophile-bearing cyclitols, including epoxides, cyclic sulfates, and a diverse set of N-alkyl aziridines. Biochemical evaluation of their inhibitory activity against GBA1, GBA2, GBA3 and GCS revealed that several N-alkyl aziridines are highly selective, nanomolar GBA2 inhibitors. Lead compounds also demonstrated metabolic stability in murine plasma, robust cellular activity and high selectivity in living cells, with no detectable GCS inhibition. Notably, octyl-alkyne aziridine 5 selectively inhibited GBA2 in mouse brain, representing the first covalent GBA2 inhibitor with demonstrated in vivo activity and brain permeability. Together, these results establish β-d-Araf cyclitol aziridine 5 as a versatile inhibitor for selective, covalent GBA2 inhibition, well suited to dissect GBA2 biology in vivo, particularly in neurological lysosomal storage disorders.
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Sep 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Massimo
Tedeschi
,
Vincent A. J.
Lit
,
Nicholas G. S.
Mcgregor
,
Tessa
Gote
,
Prajeesh
Kooloth Valappil
,
Mark
Arentshorst
,
Bogdan I.
Florea
,
Berend
Gagestein
,
Zachary
Armstrong
,
Jeroen D. C.
Codée
,
Alba
Nin-Hill
,
Carme
Rovira
,
Arthur F. J.
Ram
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: β-Mannanases are endo-acting glycoside hydrolases (GHs) that cleave β-1,4 glycosidic linkages in mannan-rich plant cell wall polysaccharides. They find application in the food and paper industries. Activity-based probes (ABPs) are powerful tools for GH profiling in complex biological samples, yet to date, bespoke ABPs reporting on mannanases have not been reported. Here, we describe the synthesis of cyclophellitol-inspired ABPs based on mannobiose, mannotriose, and glucomannose, and their use in reporting mannanase activities in secretomes from saprophytic bacteria and fungi grown on mannan-containing biomass polysaccharides. In addition to mannanases, our ABPs also labelled cellulases in secretomes from both Aspergillus niger and Cellvibrio japonicus, which may indicate broader (“negative-subsite”) substrate specificity in these enzymes. Mechanistic proof of active-site nucleophile labelling by our ABPs was obtained for both AnManA and CjMan26C by X-ray crystallography and for both AnManA and AnMan26A by mass spectrometry. Together, our results establish mannanase-targeted ABPs that may find use alongside existing reagents that report on retaining GHs that process other bulk polysaccharides.
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Jul 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Open Access
Abstract: Xylanases are central to lignocellulosic biomass degradation, yet current methods lack the specificity to resolve how enzymes distinguish complex xylan structures decorated with arabinofuranose (Araf) and 4-O-methyl-glucuronic acid (MeGlcA). Here, we report a suite of chemically-defined activity-based probes (ABPs) that enable the selective detection of arabinoxylan- and glucuronoxylan-specific xylanases (AXXs and GXXs). These cyclophellitol-derived ABPs covalently label retaining xylanases at their active sites, allowing precise mapping of substrate specificity across diverse glycoside hydrolase families. Crystallographic and mass spectrometric analyses reveal the molecular basis of probe selectivity, while in-gel and pull-down assays demonstrate their effectiveness in profiling xylanase activities in complex bacterial and fungal proteomes, including cellulosomes. By integrating activity-based protein profiling (ABPP) with sequence similarity networks (SSNs), we further show that xylanase specificity can be predicted from sequence alone, enabling rapid functional annotation of uncharacterized xylanases. This chemoproteomic strategy provides a powerful platform for discovering and engineering substrate-specific enzymes for biomass valorisation, microbial ecology, and biotechnological applications.
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Jun 2026
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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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