I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31229]
Abstract: L-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler (SW) pathway is the primary route for AsA biosynthesis and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focusses on three of them from the model system Myrciaria dubia (camu-camu): GDP-D-mannose 3',5'-epimerase (GME), L-galactose dehydrogenase (L-GalDH), and L-galactono-1,4-lactone dehydrogenase (L-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. MdL-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue from spinach. Finally, the structure of MdL-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with the FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants and the information provided should prove useful for biotechnological applications.
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Jan 2024
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I13-1-Coherence
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Quan
Wan
,
Nicholas T. H.
Farr
,
Peng
Li
,
Darren
Batey
,
Christoph
Rau
,
John
Rodenburg
,
Leihao
Lu
,
Peter R.
Laity
,
Zongpu
Xu
,
Chris
Holland
,
Cornelia
Rodenburg
,
Mingying
Yang
Diamond Proposal Number(s):
[28729]
Open Access
Abstract: Biologically derived hierarchical structural materials not only boast energy-efficient processing but also exhibit impressive mechanical performance. Silk stands as the gold standard in hierarchical fiber production, leveraging a unique combination of advantages. Nevertheless, the artificial replication of silk poses technical challenges related to precision processing and comprehensive molecular control. To address such issues, this study investigates the hierarchical assembly of solid regenerated silk in an air atmosphere, facilitated by the incorporation of carbon nanotube (CNT) seeding. Results obtained highlight that this CNT seeding facilitates multiscale structure development in response to post-spin tensile stress. Such CNT bridged structure assembly bypasses some natural processing control variables (pH, ions) and the necessary solvent immersed state for conventional silk post-drawing. Combining secondary electron hyperspectral imaging and 3D synchrotron X-ray ptychotomography, this study reports silk protein conversion from a disordered as-spun state to a longitudinal orientated semi-crystalline nano structure during drawing. The development of microscale structure during the drawing process is attributed to the presence of CNTs, yielding mechanical properties comparable to, and frequently surpassing, those exhibited by native fibers. These findings collectively propose a framework for exploring novel processing routes and offer a practical means controlling self-assembly in silk materials.
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Jan 2024
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15705]
Abstract: Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.
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Jan 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108]
Open Access
Abstract: In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05–Rpn10vWA complex at 2.17 Å resolution and of the SAP05–SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a “loop surface” with three protruding loops that form electrostatic interactions with ZnF, and a “sheet surface” featuring two β-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin–proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.
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Dec 2023
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I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[28477]
Abstract: Dry plant matter (biomass) is an abundantly available raw material for the production of biofuels. The principal carbohydrate polymer it contains, cellulose, is packed with glucose units that can be fermented into bioethanol - a sustainable liquid fuel. These polymers are difficult to break down chemically, but we get a helping hand from the natural enzymes that have evolved to do the job. Widely found enzymes, lytic polysaccharide monooxygenases (LPMOs), are major contributors to natural carbon recycling and are now used in commercial bioethanol production. However, questions remain around how these enzymes survive the powerful chemistry they wield. In work recently published in the Journal of the American Chemical Society, researchers from the University of Manchester, Novozymes, Graz University of Technology, the University of York and Diamond Light Source, used a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, electron paramagnetic resonance spectroscopy and High Energy Resolution Fluorescence Detection X-ray Absorption Spectroscopy (HERFD−XAS) to investigate how these oxidative enzymes protect themselves from harmful side reactions. Their results show that short-lived molecules produced during the breakdown of polysaccharides provide a built-in defence and repair mechanism.
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Dec 2023
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I03-Macromolecular Crystallography
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Alexandra
Males
,
Ken
Kok
,
Alba
Nin-Hill
,
Nicky
De Koster
,
Sija
Van Den Beukel
,
Thomas J. M.
Beenakker
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Herman S.
Overkleeft
,
Carme
Rovira
,
Gideon J.
Davies
,
Marta
Artola
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 → 3H4‡ → 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific “isoform-dependent” inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-D-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a “bump”, in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-D-mannosidase inhibition through a bump-and-hole approach.
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Nov 2023
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I04-Macromolecular Crystallography
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George H.
Hutchins
,
Sebastian
Kiehstaller
,
Pascal
Poc
,
Abigail H.
Lewis
,
Jisun
Oh
,
Raya
Sadighi
,
Nicholas M.
Pearce
,
Mohamed
Ibrahim
,
Ivana
Drienovská
,
Anouk M.
Rijs
,
Saskia
Neubacher
,
Sven
Hennig
,
Tom N.
Grossmann
Diamond Proposal Number(s):
[25413]
Open Access
Abstract: Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.
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Nov 2023
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[29072]
Open Access
Abstract: Synthetic cells, like their biological counterparts, require internal compartments with distinct chemical and physical properties where different functionalities can be localized. Inspired by membrane-less compartmentalization in biological cells, here, we demonstrate how microphase separation can be used to engineer heterogeneous cell-like architectures with programmable morphology and compartment-targeted activity. The synthetic cells self-assemble from amphiphilic DNA nanostructures, producing core-shell condensates due to size-induced de-mixing. Lipid deposition and phase-selective etching are then used to generate a porous pseudo-membrane, a cytoplasm analog, and membrane-less organelles. The synthetic cells can sustain RNA synthesis via in vitro transcription, leading to cytoplasm and pseudo-membrane expansion caused by an accumulation of the transcript. Our approach exemplifies how architectural and functional complexity can emerge from a limited number of distinct building blocks, if molecular-scale programmability, emergent biophysical phenomena, and biochemical activity are coupled to mimic those observed in live cells.
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Nov 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32544]
Open Access
Abstract: Fructosyl peptide oxidases (FPOX) are deglycating enzymes that find application as key enzymatic components in diabetes monitoring devices. Indeed, their use with blood samples can provide a measurement of the concentration of glycated hemoglobin and glycated albumin, two well-known diabetes markers. However, the FPOX currently employed in enzymatic assays cannot directly detect whole glycated proteins, making it necessary to perform a preliminary proteolytic treatment of the target protein to generate small glycated peptides that can act as viable substrates for the enzyme. This is a costly and time consuming step. In this work, we used an in silico protein engineering approach to enhance the overall thermal stability of the enzyme and to improve its catalytic activity toward large substrates. The final design shows a marked improvement in thermal stability relative to the wild type enzyme, a distinct widening of its access tunnel and significant enzymatic activity towards a range of glycated substrates.
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Oct 2023
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I20-Scanning-X-ray spectroscopy (XAS/XES)
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Jingming
Zhao
,
Ying
Zhuo
,
Daniel E.
Diaz
,
Muralidharan
Shanmugam
,
Abbey J.
Telfer
,
Peter J.
Lindley
,
Daniel
Kracher
,
Takahiro
Hayashi
,
Lisa S.
Seibt
,
Florence J.
Hardy
,
Oliver
Manners
,
Tobias M.
Hedison
,
Katherine A.
Hollywood
,
Reynard
Spiess
,
Kathleen M.
Cain
,
Sofia
Diaz-Moreno
,
Nigel S.
Scrutton
,
Morten
Tovborg
,
Paul H.
Walton
,
Derren J.
Heyes
,
Anthony P.
Green
Diamond Proposal Number(s):
[28477]
Open Access
Abstract: Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII–(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII–tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues “on the fly” to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
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Sep 2023
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