I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[28511]
Open Access
Abstract: The contents of biological cells are retained within compartments formed of phospholipid membranes. The movement of material within and between cells is often mediated by the fusion of phospholipid membranes, which allows mixing of contents or excretion of material into the surrounding environment. Biological membrane fusion is a highly regulated process that is catalyzed by proteins and often triggered by cellular signaling. In contrast, the controlled fusion of polymer-based membranes is largely unexplored, despite the potential application of this process in nanomedicine, smart materials, and reagent trafficking. Here, we demonstrate triggered polymersome fusion. Out-of-equilibrium polymersomes were formed by ring-opening metathesis polymerization-induced self-assembly and persist until a specific chemical signal (pH change) triggers their fusion. Characterization of polymersomes was performed by a variety of techniques, including dynamic light scattering, dry-state/cryogenic-transmission electron microscopy, and small-angle X-ray scattering (SAXS). The fusion process was followed by time-resolved SAXS analysis. Developing elementary methods of communication between polymersomes, such as fusion, will prove essential for emulating life-like behaviors in synthetic nanotechnology.
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Mar 2023
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I03-Macromolecular Crystallography
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Open Access
Abstract: Microbes that have evolved to live on lignocellulosic biomass face unique challenges in the effective and efficient use of this material as food. The bacterium Shewanella sp. ANA-3 has the potential to utilize arabinan and arabinoxylan, and uptake of the monosaccharide, l-arabinose, derived from these polymers, is known to be mediated by a single ABC transporter. We demonstrate that the substrate binding protein of this system, GafASw, binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafASw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafAEc) revealed binding site adaptations that confer specificity for furanose over pyranose forms of monosaccharides, while selecting arabinose over another related monosaccharide, galactose. The discovery of a bacterium with a natural predilection for a sugar found abundantly in certain lignocellulosic materials suggests an intimate connection in the enzymatic release and uptake of the sugar, perhaps to prevent other microbes scavenging this nutrient before it mutarotates to l-arabinopyranose. This biological discovery also provides a clear route to engineer more efficient utilization of plant biomass components in industrial biotechnology.
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Mar 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Luisana
Avilan
,
Bruce R.
Lichtenstein
,
Gerhard
Koenig
,
Michael
Zahn
,
Mark D.
Allen
,
Liliana
Oliveira
,
Matilda
Clark
,
Victoria
Bemmer
,
Rosie
Graham
,
Harry P.
Austin
,
Graham
Dominick
,
Christopher W.
Johnson
,
Gregg T.
Beckham
,
John
Mcgeehan
,
Andrew R.
Pickford
Diamond Proposal Number(s):
[17212]
Abstract: Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. Here, we report that this inhibition is dependent on incubation time, the solution conditions and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which our simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis, and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.
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Feb 2023
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I03-Macromolecular Crystallography
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Kevin J.
Mcnaught
,
Eugene
Kuatsjah
,
Michael
Zahn
,
Érica T.
Prates
,
Huiling
Shao
,
Gayle J.
Bentley
,
Andrew R.
Pickford
,
Josephine N.
Gruber
,
Kelley V.
Hestmark
,
Daniel A.
Jacobson
,
Brenton C.
Poirier
,
Chen
Ling
,
Myrsini
San Marchi
,
William E.
Michener
,
Carrie D.
Nicora
,
Jacob N.
Sanders
,
Caralyn J.
Szostkiewicz
,
Dušan
Veličković
,
Mowei
Zhou
,
Nathalie
Munoz
,
Young-Mo
Kim
,
Jon K.
Magnuson
,
Kristin E.
Burnum-Johnson
,
Kendall N.
Houk
,
John E.
Mcgeehan
,
Christopher W.
Johnson
,
Gregg T.
Beckham
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modelling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
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Feb 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I23-Long wavelength MX
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Eugene
Kuatsjah
,
Michael
Zahn
,
Xiangyang
Chen
,
Ryo
Kato
,
Daniel J.
Hinchen
,
Mikhail O.
Konev
,
Rui
Katahira
,
Christian
Orr
,
Armin
Wagner
,
Yike
Zou
,
Stefan J.
Haugen
,
Kelsey J.
Ramirez
,
Joshua K.
Michener
,
Andrew R.
Pickford
,
Naofumi
Kamimura
,
Eiji
Masai
,
Kendall N.
Houk
,
John
Mcgeehan
,
Gregg T.
Beckham
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
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Jan 2023
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I03-Macromolecular Crystallography
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Andrius
Jasilionis
,
Magdalena
Plotka
,
Lei
Wang
,
Sebastian
Dorawa
,
Joanna
Lange
,
Hildegard
Watzlawick
,
Tom
Van Den Bergh
,
Bas
Vroling
,
Josef
Altenbuchner
,
Anna-Karina
Kaczorowska
,
Ehmke
Pohl
,
Tadeusz
Kaczorowski
,
Eva
Nordberg Karlsson
,
Stefanie
Freitag-Pohl
Diamond Proposal Number(s):
[18598]
Abstract: Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared to protein structures of similar proteins. The functional characterisation of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity towards thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterised with a temperature optimum at 85 °C. The extraordinary high melting temperature Tm 102.6 °C confirms fold stability up to approximately 100 °C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.
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Jan 2023
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I03-Macromolecular Crystallography
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Erika
Erickson
,
Japheth E.
Gado
,
Luisana
Avilán
,
Felicia
Bratti
,
Richard K.
Brizendine
,
Paul A.
Cox
,
Raj
Gill
,
Rosie
Graham
,
Dong-Jin
Kim
,
Gerhard
König
,
William E.
Michener
,
Saroj
Poudel
,
Kelsey J.
Ramirez
,
Thomas J.
Shakespeare
,
Michael
Zahn
,
Eric S.
Boyd
,
Christina M.
Payne
,
Jennifer L.
Dubois
,
Andrew R.
Pickford
,
Gregg T.
Beckham
,
John E.
Mcgeehan
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
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Dec 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29835]
Open Access
Abstract: Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. We present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a “flipped-out” conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. In this work, we reveal the molecular basis of antibody recognition by bacterial enzymes, providing a template for the development of next-generation enzymes.
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Dec 2022
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B18-Core EXAFS
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Open Access
Abstract: Strontium-90 is a radionuclide of concern that is mobile in soil and groundwater and is a threat to life. Activated hydrogel biopolymer composites were fabricated for strontium remediation from groundwater. Batch uptake demonstrated a maximal stontium uptake capacity of 109 mg g−1, much higher than unactivated hydrogel controls. Activation also more than doubled the decontamination factor at environmentally relevant concentrations. EXAFS was used to investigate the binding mechanism, revealing inner sphere complexation of strontium for the first time. Biopolymer composities synthesized for these studies are sustainable and cheap remediation materials that exhibit good strontium uptake and inner sphere binding.
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Nov 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28394]
Open Access
Abstract: Antifungal proteins (AFPs) are promising antimicrobial compounds that represent a feasible alternative to fungicides. Penicillium expansum encodes three phylogenetically distinct AFPs (PeAfpA, PeAfpB and PeAfpC) which show different antifungal profiles and fruit protection effects. To gain knowledge about the structural determinants governing their activity, we solved the crystal structure of PeAfpB and rationally designed five PeAfpA::PeAfpB chimeras (chPeAFPV1-V5). Chimeras showed significant differences in their antifungal activity. chPeAFPV1 and chPeAFPV2 improved the parental PeAfpB potency, and it was very similar to that of PeAfpA. chPeAFPV4 and chPeAFPV5 showed an intermediate profile of activity compared to the parental proteins while chPeAFPV3 was inactive towards most of the fungi tested. Structural analysis of the chimeras evidenced an identical scaffold to PeAfpB, suggesting that the differences in activity are due to the contributions of specific residues and not to induced conformational changes or structural rearrangements. Results suggest that mannoproteins determine protein interaction with the cell wall and its antifungal activity while there is not a direct correlation between binding to membrane phospholipids and activity. This work provides new insights about the relevance of sequence motifs and the feasibility of modifying protein specificity, opening the door to the rational design of chimeras with biotechnological applicability.
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Nov 2022
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