I02-Macromolecular Crystallography
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Diana O.
Ribeiro
,
Aldino
Viegas
,
Virgínia M. R.
Pires
,
João
Medeiros‐silva
,
Pedro
Bule
,
Wengang
Chai
,
Filipa
Marcelo
,
Carlos M. G. A.
Fontes
,
Eurico J.
Cabrita
,
Angelina S.
Palma
,
Ana Luisa
Carvalho
Diamond Proposal Number(s):
[16609]
Abstract: Understanding the specific molecular interactions between proteins and β1,3‐1,4‐mixed‐linked d‐glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate‐binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3‐1,4‐mixed‐linked over β1,4‐linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3‐1,4‐linked glucose oligosaccharides showed that both the chain length and the position of the β1,3‐linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site‐directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3‐1,4‐mixed‐linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH‐π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3‐linkage at the reducing end and at specific positions along the β1,4‐linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed‐linked β‐glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture.
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Dec 2019
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I02-Macromolecular Crystallography
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Christian
Roth
,
Olga V.
Moroz
,
Johan P.
Turkenburg
,
Elena
Blagova
,
Jitka
Waterman
,
Antonio
Ariza
,
Li
Ming
,
Sun
Tianqi
,
Carsten
Andersen
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[1221, 9948]
Open Access
Abstract: Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.
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Oct 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Transaminases are pyridoxal-5′-phosphate (PLP) binding enzymes, broadly studied for their potential industrial application. Their affinity for PLP has been related to their performance and operational stability and while significant differences in PLP requirements have been reported, the environment of the PLP-binding pocket is highly conserved. In this study, thorough analysis of the residue interaction network of three homologous transaminases Halomonas elongata (HeTA), Chromobacterium violaceum (CvTA), and Pseudomonas fluorescens (PfTA) revealed a single residue difference in their PLP binding pocket: an asparagine at position 120 in HeTA. N120 is suitably positioned to interact with an aspartic acid known to protonate the PLP pyridinium nitrogen, while the equivalent position is occupied by a valine in the other two enzymes. Three different mutants were constructed (HeTA-N120V, CvTA-V124N, and PfTA-V129N) and functionally analyzed. Notably, in HeTA and CvTA, the asparagine variants, consistently exhibited a higher thermal stability and a significant decrease in the dissociation constant (Kd) for PLP, confirming the important role of N120 in PLP binding. Moreover, the reaction intermediate pyridoxamine-5′-phosphate (PMP) was released more slowly into the bulk, indicating that the mutation also enhances their PMP binding capacity. The crystal structure of PfTA, elucidated in this work, revealed a tetrameric arrangement with the PLP binding sites near the subunit interface. In this case, the V129N mutation had a negligible effect on PLP-binding, but it reduced its temperature stability possibly destabilizing the quaternary structure.
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Oct 2019
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
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Open Access
Abstract: Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.
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Sep 2019
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[15771]
Abstract: This research aimed to find the best phenotype of the brown algae Fucus vesiculosus (kelp) which has the greater potential to become a sorption byproduct for Zn removal from contaminated waters. Thus, the Zn uptake capacity and sorption mechanisms of the kelp collected from the Baltic Sea shore was, for the first time, investigated under various conditions, and compared to the phenotype habiting on the Irish Sea shore. Sorption studies were performed investigating the effect of algal dosage, Zn sources as well as algal harvesting time of the year on Zn uptake capacity. The results suggested that the Baltic algae is a better biosorbent for Zn uptake. Sorption mechanisms were studied by employing various indirect and direct approaches, more importantly, including high resolution synchrotron X-Ray Fluorescence and X-Ray Absorption Spectroscopy (XAS) and molecular modelling (MM). The results revealed that alginate and cellulose are among the main polysaccharide bonding Zn at algal surface, via coordination with O atoms from carboxyl and hydroxyl groups. XAS results giving direct measurements of Zn bonding environment on algal surface are supported by MM outputs and suggested that Zn is surrounded by ca. 5 O atoms at interatomic distances varying from 1.94 to 2.02 Å. The results contribute to understanding sorption mechanisms which can further lead to finding the best eluent for Zn desorption from the used biomass, bio sorbent reconditioning and reuse in multiple sorption desorption cycles as well as process optimization before industrial scaling up.
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Aug 2019
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B21-High Throughput SAXS
B23-Circular Dichroism
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Wenjin
Xiao
,
Thomas I. P.
Green
,
Xiaowen
Liang
,
Rosalia Cuahtecontzi
Delint
,
Guillaume
Perry
,
Michael S.
Roberts
,
Kristian
Le Vay
,
Catherine R.
Back
,
Raimomdo
Ascione
,
Haolu
Wang
,
Paul R.
Race
,
Adam W.
Perriman
Open Access
Abstract: We present a new cell membrane modification methodology where the inherent heart tissue homing properties of the infectious bacteria Streptococcus gordonii are transferred to human stem cells. This is achieved via the rational design of a chimeric protein–polymer surfactant cell membrane binding construct, comprising the cardiac fibronectin (Fn) binding domain of the bacterial adhesin protein CshA fused to a supercharged protein. Significantly, the protein–polymer surfactant hybrid spontaneously inserts into the plasma membrane of stem cells without cytotoxicity, instilling the cells with a high affinity for immobilized fibronectin. Moreover, we show that this cell membrane reengineering approach significantly improves retention and homing of stem cells delivered either intracardially or intravenously to the myocardium in a mouse model.
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Jul 2019
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[20975]
Open Access
Abstract: Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.
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Jul 2019
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I04-Macromolecular Crystallography
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Melodie M.
Machovina
,
Sam J. B.
Mallinson
,
Brandon C.
Knott
,
Alexander W.
Meyers
,
Marc
Garcia-Borràs
,
Lintao
Bu
,
Japheth E.
Gado
,
April
Oliver
,
Graham P.
Schmidt
,
Daniel J.
Hinchen
,
Michael F.
Crowley
,
Christopher W.
Johnson
,
Ellen L.
Neidle
,
Christina M.
Payne
,
Kendall N.
Houk
,
Gregg T.
Beckham
,
John E.
Mcgeehan
,
Jennifer L.
Dubois
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.
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Jun 2019
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[21755]
Abstract: We show that the azamacrocycle ‘cyclam’ (1,4,8,11-tetraazacyclodecane) in conjunction with a silicon catecholate ion generates novel hollow tetragonal tube-like crystalline materials [(C6H4O2)3Si][C10H26N4]·H2O, whose dimensions can be tuned according to the pH of the reaction medium. The synthesis approach was successful for both silicon and germanium and we hypothesise that a range of other catecholate precursors of elements such as iron could be used to generate a large array of inorganic materials with interesting morphologies. The synthesis approach can be extended to tertiary diamines with functional group spacing playing an important role in the efficacy of complexation. Of the molecules explored to date, a C2 spacing (N,N,N′,N′-tetramethylethylenediamine (4MEDAE)), leads to the most efficient structure control with hollow hexagonal tube-like structures being formed. In addition, we show that azamacrocycles, in the presence of unbuffered tetramethoxysilane (TMOS) solutions can be used to manipulate silica formation and provide a fast (ca. 10 minutes) synthesis route to particles whose diameter can be tuned from ca. 20 nm to several hundreds of nm under reaction conditions (no extremes of pH) that make the sols suitable for direct use in biotechnological applications.
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Jun 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Kalyanasundaram
Subramanian
,
Karolina
Mitusińska
,
John
Raedts
,
Feras
Almourfi
,
Henk-Jan
Joosten
,
Sjon
Hendricks
,
Svetlana E.
Sedelnikova
,
Servé W. M.
Kengen
,
Wilfred R.
Hagen
,
Artur
Góra
,
Vitor A. P. Martins
Dos Santos
,
Patrick
Baker
,
John
Van Der Oost
,
Peter J.
Schaap
Diamond Proposal Number(s):
[1218]
Open Access
Abstract: The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.
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May 2019
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