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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Krishnan
Srinivas
,
Amelia K.
Gilio
,
Mahima
Sharma
,
Lawrence
Green
,
Alexander
Ascham
,
Jack
Domenech
,
Balázs
Pogrányi
,
Jiacheng
Li
,
Scott P.
France
,
Russell D.
Lewis
,
William P.
Unsworth
,
Gideon
Grogan
Diamond Proposal Number(s):
[24948]
Abstract: Imine reductases with an (S)-preference for the reduction of the model substrate 2-methyl pyrroline typically contain tyrosine in the active site (Y-IREDs) instead of the aspartate present within (R)-selective enzymes (D-IREDs). As with D-IREDs, a subset of Y-IREDs is capable of enabling reductive amination reactions between some ketone and amine partners to give optically active amines with high optical purity. However, structures of Y-IREDs in complex with the substrates and products of the reductive amination have not been forthcoming. Herein, structures of the Y-IRED IR91 from Kribbella flavida in complex with 5-methoxy-2-tetralone, a synthetic precursor to the anti-Parkinson's treatment rotigotine, and also its reductive amination product with methylamine, 5-methoxy-(S)-2-(N-methylamino)-tetralin, are presented. The structures, in combination with mutation and kinetic studies, support a role for tryptophan residue W258 in the activity of the enzyme, possibly in binding of the ketone prior to reaction with methylamine.
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Jul 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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Diamond Proposal Number(s):
[24948, 18598]
Open Access
Abstract: The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY-family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol-aziridine activity-based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol-aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5-probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin-probe enables SQase capture and identification by proteomics. The Cy5-probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT-qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.
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Apr 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Abstract: The solute-binding protein (SBP) components of periplasmic binding protein-dependent ATP-binding cassette (ABC)-type transporters often possess exquisite selectivity for their cognate ligands. Maltose binding protein (MBP), the best studied of these SBPs, has been extensively used as a fusion partner to enable the affinity purification of recombinant proteins. However, other SBPs and SBP-ligand based affinity systems remain underexplored. The sulfoquinovose-binding protein SmoF, is a substrate-binding protein component of the ABC transporter cassette in Agrobacterium tumefaciens involved in importing sulfoquinovose (SQ) and its derivatives for SQ catabolism. Here, we show that SmoF binds with high affinity to the octyl glycoside of SQ (octyl-SQ), demonstrating remarkable tolerance to extension of the anomeric substituent. The 3D X-ray structure of the SmoF·octyl-SQ complex reveals accommodation of the octyl chain, which projects to the protein surface, providing impetus for the synthesis of a linker-equipped SQ-amine using a thiol–ene reaction as a key step, and its conjugation to cyanogen bromide modified agarose. We demonstrate the successful capture and release of SmoF from SQ-agarose resin using SQ as competitive eluant, and selectivity for release versus other organosulfonates. We show that SmoF can be captured and purified from a cell lysate, demonstrating the utility of SQ-agarose in capturing SQ binding proteins from complex mixtures. The present work provides a pathway for development of ‘capture-and-release’ affinity resins for the discovery and study of SBPs.
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Mar 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Open Access
Abstract: The sulfosugar sulfoquinovose (SQ) is produced by photosynthetic plants, algae, and cyanobacteria on a scale of 10 billion tons per annum. Its degradation, which is essential to allow cycling of its constituent carbon and sulfur, involves specialized glycosidases termed sulfoquinovosidases (SQases), which release SQ from sulfolipid glycoconjugates, so SQ can enter catabolism pathways. However, many SQ catabolic gene clusters lack a gene encoding a classical SQase. Here, we report the discovery of a new family of SQases that use an atypical oxidoreductive mechanism involving NAD+ as a catalytic cofactor. Three-dimensional X-ray structures of complexes with SQ and NAD+ provide insight into the catalytic mechanism, which involves transient oxidation at C3. Bioinformatic survey reveals this new family of NAD+-dependent SQases occurs within sulfoglycolytic and sulfolytic gene clusters that lack classical SQases and is distributed widely including within Roseobacter clade bacteria, suggesting an important contribution to marine sulfur cycling.
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Dec 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598, 24948]
Open Access
Abstract: Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas (sulfo-EMP) pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product DHAP reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate binding pocket, two metal binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfo-EMP gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases occur exclusively within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.
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Oct 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Å resolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Å resolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.
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Sep 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Sulfoquinovose (SQ) is a key component of plant sulfolipids (sulfoquinovosyl diacylglycerols) and a major environmental reservoir of biological sulfur. Breakdown of SQ is achieved by bacteria through the pathways of sulfoglycolysis. The sulfoglycolytic sulfofructose transaldolase (sulfo-SFT) pathway is used by gut-resident firmicutes and soil saprophytes. After isomerization of SQ to sulfofructose (SF), the namesake enzyme catalyzes the transaldol reaction of SF transferring dihydroxyacetone to 3C/4C acceptors to give sulfolactaldehyde and fructose-6-phosphate or sedoheptulose-7-phosphate. We report the 3D cryo-EM structure of SF transaldolase from Bacillus megaterium in apo and ligand bound forms, revealing a decameric structure formed from two pentameric rings of the protomer. We demonstrate a covalent “Schiff base” intermediate formed by reaction of SF with Lys89 within a conserved Asp-Lys-Glu catalytic triad and defined by an Arg-Trp-Arg sulfonate recognition triad. The structural characterization of the signature enzyme of the sulfo-SFT pathway provides key insights into molecular recognition of the sulfonate group of sulfosugars.
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Feb 2023
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