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
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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
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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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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I03-Macromolecular Crystallography
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Yu
Liu
,
Ganka
Bineva-Todd
,
Richard W.
Meek
,
Laura
Mazo
,
Beatriz
Piniello
,
Olga
Moroz
,
Sean A.
Burnap
,
Nadima
Begum
,
André
Ohara
,
Chloe
Roustan
,
Sara
Tomita
,
Svend
Kjaer
,
Karen
Polizzi
,
Weston B.
Struwe
,
Carme
Rovira
,
Gideon J.
Davies
,
Benjamin
Schumann
Diamond Proposal Number(s):
[32736]
Open Access
Abstract: Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme–substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.
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Oct 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sandeep
Bhosale
,
Sachin
Kandalkar
,
Pierre-André
Gilormini
,
Oluwafemi
Akintola
,
Rhianna
Rowland
,
Pal John Pal
Adabala
,
Dustin
King
,
Matthew C.
Deen
,
Xi
Chen
,
Gideon J.
Davies
,
David J.
Vocadlo
,
Andrew J.
Bennet
Diamond Proposal Number(s):
[18598, 24948]
Open Access
Abstract: Mutations in many members of the set of human lysosomal glycoside hydrolases cause a wide range of lysosomal storage diseases. As a result, much effort has been directed toward identifying pharmacological chaperones of these lysosomal enzymes. The majority of the candidate chaperones are active site-directed competitive iminosugar inhibitors but these have met with limited success. As a first step toward an alternative class of pharmacological chaperones we explored the potential of small molecule mechanism-based reversible covalent inhibitors to form transient enzyme–inhibitor adducts. By serial synthesis and kinetic analysis of candidate molecules, we show that rational tuning of the chemical reactivity of glucose-configured carbasugars delivers cyclohexenyl-based allylic carbasugar that react with the lysosomal enzyme β-glucocerebrosidase (GCase) to form covalent enzyme-adducts with different half-lives. X-ray structural analysis of these compounds bound noncovalently to GCase, along with the structures of the covalent adducts of compounds that reacted with the catalytic nucleophile of GCase, reveal unexpected reactivities of these compounds. Using differential scanning fluorimetry, we show that formation of a transient covalent intermediate stabilizes the folded enzyme against thermal denaturation. In addition, these covalent adducts break down to liberate the active enzyme and a product that is no longer inhibitory. We further show that the one compound, which reacts through an unprecedented SN1′-like mechanism, exhibits exceptional reactivity–illustrated by this compound also covalently labeling an α-glucosidase. We anticipate that such carbasugar-based single turnover covalent ligands may serve as pharmacological chaperones for lysosomal glycoside hydrolases and other disease-associated retaining glycosidases. The unusual reactivity of these molecules should also open the door to creation of new chemical biology probes to explore the biology of this important superfamily of glycoside hydrolases.
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Sep 2024
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