B18-Core EXAFS
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Diamond Proposal Number(s):
[17052]
Abstract: Hydrogen peroxide is a co-substrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic poly-saccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high but it is accompa-nied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV/vis, CD, XAS, EPR, VT/VH-MCD and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)-tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)-tyrosyl radical center entails the formation of significant Cu(II)-(●OTyr) overlap, which in turn requires that the plane of the d(x2-y2) SOMO of the Cu(II) is orientated towards the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a ‘hole-hopping’ charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.
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Nov 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Brønsted acid and Asp as a Brønsted base in a single-displacement mechanism. A predominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms.
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Oct 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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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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M. Fleur
Sernee
,
Julie E.
Ralton
,
Tracy L.
Nero
,
Lukasz F.
Sobala
,
Joachim
Kloehn
,
Marcel A.
Vieira-lara
,
Simon A.
Cobbold
,
Lauren
Stanton
,
Douglas E. V.
Pires
,
Eric
Hanssen
,
Alexandra
Males
,
Tom
Ward
,
Laurence M.
Bastidas
,
Phillip L.
Van Der Peet
,
Michael W.
Parker
,
David B.
Ascher
,
Spencer J.
Williams
,
Gideon J.
Davies
,
Malcolm J.
Mcconville
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.
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Sep 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Background: The quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate.
Results: Although classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a “histidine-brace” catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases.
Conclusion: Shipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.
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Sep 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Enzyme transition-state mimics can act as powerful inhibitors and allow structural studies that report on the conformation of the transition-state. Here, mannoimidazole, a mimic of the transition state of mannosidase catalyzed hydrolysis of mannosides, is shown to bind in a B2,5 conformation on the Clostridium perfringens GH125 α-1,6-mannosidase, providing additional evidence of a OS2–B2,5–1S5 conformational itinerary for enzymes of this family.
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Aug 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Marta
Artola
,
Christinne
Hedberg
,
Rhianna J.
Rowland
,
Lluís
Raich
,
Kassiani
Kytidou
,
Liang
Wu
,
Amanda
Schaaf
,
Maria Joao
Ferraz
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Carme
Rovira
,
Johannes M. F. G.
Aerts
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Fabry disease is an inherited lysosomal storage disorder that is characterized by a deficiency in lysosomal α-D-galactosidase activity. One current therapeutic strategy involves enzyme replacement therapy, in which patients are treated with a recombinant enzyme. Co-treatment with enzyme active-site stabilizers is advocated to increase treatment efficacy, a strategy that requires effective and selective enzyme stabilizers. Here, we describe the design and development of an α-D-gal-cyclophellitol cyclosulfamidate as a new class of neutral, conformationally constrained competitive glycosidase inhibitors that act by mimicry of the Michaelis complex conformation. We found that D-galactose-configured α-cyclosulfamidate 4 effectively stabilizes recombinant human α-D-galactosidase (agalsidase beta, Fabrazyme®) both in vitro and in cellulo.
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Aug 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Casper
De Boer
,
Nicholas G. S.
Mcgregor
,
Rhianna J.
Rowland
,
Olga
Moroz
,
Elena
Blagova
,
Jos
Reijngoud
,
Mark
Arentshorst
,
David
Osborn
,
Marc D.
Morant
,
Eric
Abbate
,
Mary A.
Stringer
,
Kristian B. R. M.
Krogh
,
Lluís
Raich
,
Carme
Rovira
,
Jean-guy
Berrin
,
Gilles P.
Van Wezel
,
Arthur F. J.
Ram
,
Bogdan I.
Florea
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Keith S.
Wilson
,
Liang
Wu
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Abstract: Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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May 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Gaucher disease is caused by inherited deficiency in glucocerebrosidase (GBA, a retaining β-glucosidase), and deficiency in GBA constitutes the largest known genetic risk factor for Parkinson’s disease. In the past, animal models of Gaucher disease have been generated by treatment with the mechanism-based GBA inhibitors, conduritol B epoxide (CBE), and cyclophellitol. Both compounds, however, also target other retaining glycosidases, rendering generation and interpretation of such chemical knockout models complicated. Here we demonstrate that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these.
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Mar 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864, 9948]
Abstract: The opium poppy, Papaver somniferum, has been a source of medicinal alkaloids since the earliest civilizations; ca. 3400 B.C. The benzylisoquinoline alkaloid noscapine is produced commercially in P. somniferum for use as a cough suppressant and it also has potential as an anticancer compound. The first committed step in the recently elucidated noscapine biosynthetic pathway involves the conversion of scoulerine to tetrahydrocolumbamine by 9-O-methylation, catalysed by O-methyltransferase 1 (PSMT1). We demonstrate, through protein structures (obtained through rational crystal engineering at resolutions from 1.5 to 1.2Å for the engineered variants) across the reaction coordinate, how domain closure allows specific methyl transfer to generate the product. SAM-dependent methyl transfer is central to myriad natural products in plants, analysis of amino-acid sequence, now taking the three-dimensional structure of PSMT1 and low identity homologs into account, begins to shed light on the structural features that govern substrate specificity in these key, ubiquitous, plant enzymes. We propose how “gatekeeper” residues can determine acceptor regiochemistry thus allowing prediction across the wide genomic resource.
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Mar 2019
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