I03-Macromolecular Crystallography
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Nehad
Noby
,
Husam Sabah
Auhim
,
Samuel
Winter
,
Harley L.
Worthy
,
Amira M.
Embaby
,
Hesham
Saeed
,
Ahmed
Hussein
,
Christopher R.
Pudney
,
Pierre J.
Rizkallah
,
Stephen A.
Wells
,
D. Dafydd
Jones
Diamond Proposal Number(s):
[18812]
Open Access
Abstract: Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.
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Dec 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical re-engineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent post-cleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent pre-cleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the post-cleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q and E493K confirmed the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.
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Dec 2021
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B23-Circular Dichroism
I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[17972, 18006]
Open Access
Abstract: Here, we describe a facile route to the synthesis of enzymatically active highly fabricable plastics, where the enzyme is an intrinsic component of the material. This is facilitated by the formation of an electrostatically stabilized enzyme–polymer surfactant nanoconstruct, which, after lyophilization and melting, affords stable macromolecular dispersions in a wide range of organic solvents. A selection of plastics can then be co-dissolved in the dispersions, which provides a route to bespoke 3D enzyme plastic nanocomposite structures using a wide range of fabrication techniques, including melt electrowriting, casting, and piston-driven 3D printing. The resulting constructs comprising active phosphotriesterase (arPTE) readily detoxify organophosphates with persistent activity over repeated cycles and for long time periods. Moreover, we show that the protein guest molecules, such as arPTE or sfGFP, increase the compressive Young’s modulus of the plastics and that the identity of the biomolecule influences the nanomorphology and mechanical properties of the resulting materials. Overall, we demonstrate that these biologically active nanocomposite plastics are compatible with state-of-the-art 3D fabrication techniques and that the methodology could be readily applied to produce robust and on-demand smart nanomaterial structures.
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Nov 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d-xylonolactone. We have determined crystal structures of Cc XylC in complex with d-xylonolactone isomer analogues d-xylopyranose and (r)-(+)-4-hydroxy-2-pyrrolidinone at high resolution. Cc XylC has a 6-bladed β-propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe2+. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side-chains of three amino acid residues, Glu18, Asn146, and Asp196 which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe2+. The structure of Cc XylC was compared with eight other ester hydrolases of the β-propeller hydrolase family. The previously published crystal structures of other β-propeller hydrolases contain either Ca2+, Mg2+ or Zn2+ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe2+ as a catalytic metal. This could further expand our understanding of utilization of Fe2+ not only in oxidative enzymes but also in hydrolases.
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Nov 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12579]
Open Access
Abstract: The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.
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Nov 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20303]
Open Access
Abstract: CYP505A30 is a fungal, self-sufficient cytochrome P450 monooxygenase that can selectively oxyfunctionalise n-alkanes, fatty alcohols, and fatty acids. From alkanes, it produces a mixture of non-vicinal diols by two sequential hydroxylation reactions. Here we report the structure of the haem domain of CYP505A30, the first structure for a member of the CYP505 family, with dodecanoic acid bound within the active site. Overall, a high structural similarity to the related bacterial CYP102A1 was observed, despite low sequence identity (<40 %). Comparison of the active sites, however, showed a high degree of conservation with only two amino acid differences close to the haem. Stabilisation of the acid substrate in CYP505A30 also occurs, as in CYP102A1, via an arginine residue. However, compared to R47, which is situated in the β1 region of CYP102A1, R358 is located in the β3 region of CYP505A30. We furthermore created mutants to test if it is possible to rationally transfer the knowledge on active site mutations in CYP102A1 to change the regioselectivity of CYP505A30. The introduction of F93V, I334F mutations resulted in increased ω-1 (C2) regioselectivity, similar to CYP102A1 328-87, of more than 80 % for n-octane and 90 % for n-decane. Changing residues to resemble the 102A1 wildtype increased the regioselectivity towards ω-2 (C3) to over 60 % for both substrates. The knowledge gained from this study unlocks a more selective production of symmetrical non-vicinal diols from n-alkanes.
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Oct 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24447]
Open Access
Abstract: Biological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC. Here, we report the biochemical and structural characterisation of TphC in both open and TPA-bound closed conformations. This analysis demonstrates the narrow ligand specificity of TphC towards aromatic para-substituted dicarboxylates, such as TPA and closely related analogues. Further phylogenetic and genomic context analysis of the tph genes reveals homologous operons as a genetic resource for future biotechnological and metabolic engineering efforts towards circular plastic bio-economy solutions.
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Oct 2021
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[24321]
Abstract: Looking for new green and environmentally friendly bio sorbents for metal removal from polluted wastewater, the present study investigates the potential new bio sorbent for Cd(II) removal from wastewater namely, the mechanism and uptake capacity of Cd(II) by brown algae, Fucus vesiculosus from the Irish Sea. This work takes a comprehensive approach involving the combination of qualitative and quantitative information collected from macro to atomistic scale, in a direct and non-destructive manner. Our results demonstrate that Cd(II) is adsorbed on the algal surface based on carboxylic of alginate groups. Effective Cd(II) adsorption is achieved at pH conditions between 5 and 7, at which the uptake occurs rapidly (∼2 h), with increasing Cd(II) concentration. Cd maximum uptake capacity (i.e., 1.203 mmol Cd g−1 dried algae) in first adsorption cycle show superior uptake as opposed to other species. Quantitatively the bio sorbent has an increasing uptake capacity (more than two folds) in the second cycle, after metal elution and biomass surface sites functioning. Desorption of Cd(II) and the regeneration of the biomass is effectively achieved with HCl (10 mM) and EDTA (1 mM), but they can only be used for two cycles, before the efficiency decreases. Microprecipitation occurs at high pH (>9) when using NaOH as an eluent. Results from this work shed new light on understanding Cd(II) binding mechanisms on Fucus v., providing crucial information for further process optimization, pilot testing, scaling up and implementation as a clean, environmentally friendly biotechnology applied to wastewater treatments.
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Oct 2021
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B16-Test Beamline
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Diamond Proposal Number(s):
[20046]
Open Access
Abstract: In this work, novel bioinspired polyurethane (PU) scaffolds were fabricated via freeze casting for PU-based Pancreatic Ductal Adenocarcinoma (PDAC) model. In order to reproduce the tumour micro-environment that facilitates cellular kinetics, the PU scaffolds were surface modified with extracellular matrix (ECM) proteins including collagen and fibronectin (Col and FN). Synchrotron-based small- and wide-angle X-ray scattering (SAXS/WAXS) techniques were applied to probe structural evolution during in situ mechanical testing. Strains at macroscopic, nano-, and lattice scales were obtained to investigate the effects of ECM proteins and pancreatic cell activities to PU scaffolds. Significant mechanical strengthening across length scales of PU scaffolds was observed in specimens surface modified by FN. A model of stiffness modulation via enhanced interlamellar recruitment is proposed to explain the multi-scale strengthening mechanisms. Understanding multi-scale deformation mechanisms of a series of PU scaffolds opens an opportunity in developing a novel pancreatic cancer model for studying cancer evolution and predicting outcomes of drug/treatments.
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Sep 2021
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I03-Macromolecular Crystallography
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Erika
Erickson
,
Thomas J.
Shakespeare
,
Felicia
Bratti
,
Bonnie L.
Buss
,
Rosie
Graham
,
Mckenzie A.
Hawkins
,
Gerhard
König
,
William E.
Michener
,
Joel
Miscall
,
Kelsey J.
Ramirez
,
Nicholas A.
Rorrer
,
Michael
Zahn
,
Andrew R.
Pickford
,
John E.
Mcgeehan
,
Gregg
Beckham
Diamond Proposal Number(s):
[23269]
Abstract: There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, we compare the Ideonella sakaiensis PETase wild-type enzyme to a previously reported improved variant (W159H/S238F). We compare the thermostability of each enzyme and describe a 1.45 Å resolution structure of the mutant, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies show that reaction temperature has the strongest influence on performance between the two enzymes. We also show that both enzymes achieve higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, we assess the impact of product accumulation on reaction progress for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displays different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both are sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-
based polyester recycling.
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Sep 2021
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