I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29507]
Open Access
Abstract: In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.
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Jun 2022
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[2164]
Open Access
Abstract: Protein synthesis in plant crops contributes to the balance of food and fuel on our planet that influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins, 71 plant-specific rRNA modifications, and re-annotates 30 rRNA residues in the available sequence. On the protein level, isoAsp is found in position 137 of uS11, and previously believed to be universal zinc finger is missing in eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. On the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.
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May 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Sulfoquinovose (SQ) is the anionic headgroup of the ubiquitous plant sulfolipid, sulfoquinovosyl diacylglycerol (SQDG). SQDG can undergo delipidation to give sulfoquinovosyl glycerol (SQGro) and further glycoside cleavage to give SQ, which can be metabolized through microbial sulfoglycolytic pathways. Exogenous SQDG metabolites are imported into bacteria through membrane spanning transporter proteins. The recently discovered sulfoglycolytic sulfoquinovose monooxygenase (sulfo-SMO) pathway in Agrobacterium tumefaciens features a periplasmic sulfoquinovosyl glycerol binding protein, SmoF, and an ATP-binding cassette (ABC) transporter. Here, we use X-ray crystallography, differential scanning fluorimetry and isothermal titration calorimetry to study SQ glycoside recognition by SmoF. This work reveals that in addition to SQGro, SmoF can also bind SQ, a simple methyl glycoside and even a short-chain SQDG analogue. Molecular recognition of these substrates is achieved through conserved interactions with the SQ-headgroup together with more plastic interactions with the aglycones. This suggests that the solute binding protein of A. tumefaciens, and related SQ-binding proteins from other sulfoglycolytic pathways, can provide their host organisms direct access to most of the SQ metabolites known to be produced by phototrophs.
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Mar 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60-80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy) some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high resolution crystal structure suggests a model for phytate recognition which, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well-conserved in PAPhys from monocot cereals, enzymes which are characterised by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta.
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Feb 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Mahima
Sharma
,
James P.
Lingford
,
Marija
Petricevic
,
Alexander J. D.
Snow
,
Yunyang
Zhang
,
Michael A.
Järvå
,
Janice W.-Y.
Mui
,
Nichollas E.
Scott
,
Eleanor C.
Saunders
,
Runyu
Mao
,
Ruwan
Epa
,
Bruna M.
Da Silva
,
Douglas E. V.
Pires
,
David B.
Ascher
,
Malcolm J.
Mcconville
,
Gideon J.
Davies
,
Spencer J.
Williams
,
Ethan D.
Goddard-Borger
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Catabolism of sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose), the ubiquitous sulfosugar produced by photosynthetic organisms, is an important component of the biogeochemical carbon and sulfur cycles. Here, we describe a pathway for SQ degradation that involves oxidative desulfurization to release sulfite and enable utilization of the entire carbon skeleton of the sugar to support the growth of the plant pathogen Agrobacterium tumefaciens. SQ or its glycoside sulfoquinovosyl glycerol are imported into the cell by an ATP-binding cassette transporter system with an associated SQ binding protein. A sulfoquinovosidase hydrolyzes the SQ glycoside and the liberated SQ is acted on by a flavin mononucleotide-dependent sulfoquinovose monooxygenase, in concert with an NADH-dependent flavin reductase, to release sulfite and 6-oxo-glucose. An NAD(P)H-dependent oxidoreductase reduces the 6-oxo-glucose to glucose, enabling entry into primary metabolic pathways. Structural and biochemical studies provide detailed insights into the recognition of key metabolites by proteins in this pathway. Bioinformatic analyses reveal that the sulfoquinovose monooxygenase pathway is distributed across Alpha- and Betaproteobacteria and is especially prevalent within the Rhizobiales order. This strategy for SQ catabolism is distinct from previously described pathways because it enables the complete utilization of all carbons within SQ by a single organism with concomitant production of inorganic sulfite.
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Jan 2022
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I13-2-Diamond Manchester Imaging
I18-Microfocus Spectroscopy
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Sam
Keyes
,
Arjen
Van Veelen
,
Dan
Mckay Fletcher
,
Callum
Scotson
,
Nico
Koebernick
,
Chiara
Petroselli
,
Katherine
Williams
,
Siul
Ruiz
,
Laura
Cooper
,
Robbie
Mayon
,
Simon
Duncan
,
Marc
Dumont
,
Iver
Jakobsen
,
Giles
Oldroyd
,
Andrzej
Tkacz
,
Philip
Poole
,
Fred
Mosselmans
,
Camelia
Borca
,
Thomas
Huthwelker
,
David L.
Jones
,
Tiina
Roose
Open Access
Abstract: Phosphorus (P) is essential for plant growth. Arbuscular mycorrhizal fungi (AMF) aid its uptake by acquiring sources distant from roots in return for carbon. Little is known about how AMF colonise soil pore-space, and models of AMF-enhanced P-uptake are poorly validated. We used synchrotron X-ray computed tomography (SXRCT) to visualize mycorrhizas in soil, and synchrotron X-ray fluorescence (XRF/XANES) elemental mapping for phosphorus (P), sulphur (S) and aluminium (Al), in combination with modelling. We found that AMF inoculation had a suppressive effect on colonisation by other soil fungi and identified differences in structure and growth rate between hyphae of AMF and nonmycorrhizal fungi. Results showed that AMF co-locate with areas of high P and low Al, andpreferentially associate with organic-type P species in preference to Al-rich inorganic P. We discovered that AMF avoid Al-rich areas as a source of P. S-rich regions correlated with higher hyphal density and an increased organic-associated P-pool, whilst oxidized S-species were found close to AMF hyphae. Increased S oxidation close to AMF suggested the observed changes were microbiome-related. Our experimentally-validated model led to an estimate of P-uptake by AMF hyphae that is an order of magnitude lower than rates previously estimated; a result with significant implications for modelling of plant-soil-AMF interactions.
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Jan 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)–containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the β2-β3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4C–RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY–W-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.
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Dec 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Maria
Schwarz
,
Rebecca F. M.
Eno
,
Stefanie
Freitag-Pohl
,
Christopher R.
Coxon
,
Hannah E.
Straker
,
David J.
Wortley
,
David J.
Hughes
,
Glynn
Mitchell
,
Jenny
Moore
,
Ian
Cummins
,
Nawaporn
Onkokesung
,
Melissa
Brazier-Hicks
,
Robert
Edwards
,
Ehmke
Pohl
,
Patrick G.
Steel
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: The evolution and growth of multiple-herbicide resistance (MHR) in grass weeds continues to threaten global cereal production. While various processes can contribute to resistance, earlier work has identified the phi class glutathione-S-transferase (AmGSTF1) as a functional biomarker of MHR in black-grass (Alopecurus myosuroides). This study provides further insights into the role of AmGSTF1 in MHR using a combination of chemical and structural biology. Crystal structures of wild-type AmGSTF1, together with two specifically designed variants that allowed the co-crystal structure determination with glutathione and a glutathione adduct of the AmGSTF1 inhibitor 4-chloro-7-nitro-benzofurazan (NBD-Cl) were obtained. These studies demonstrated that the inhibitory activity of NBD-Cl was associated with the occlusion of the active site and the impediment of substrate binding. A search for other selective inhibitors of AmGSTF1, using ligand-fishing experiments, identified a number of flavonoids as potential ligands. Subsequent experiments using black-grass extracts discovered a specific flavonoid as a natural ligand of the recombinant enzyme. A series of related synthetic flavonoids was prepared and their binding to AmGSTF1 was investigated showing a high affinity for derivatives bearing a O-5-decyl-α-carboxylate. Molecular modelling based on high-resolution crystal structures allowed a binding pose to be defined which explained flavonoid binding specificity. Crucially, high binding affinity was linked to a reversal of the herbicide resistance phenotype in MHR black-grass. Collectively, these results present a nature-inspired new lead for the development of herbicide synergists to counteract MHR in weeds.
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Nov 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565, 13467]
Open Access
Abstract: Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.
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Nov 2021
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity.
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Oct 2021
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