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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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Olga V.
Moroz
,
Elena
Blagova
,
Verena
Reiser
,
Rakhi
Saikia
,
Sohel
Dalal
,
Christian Isak
Jørgensen
,
Vikram K.
Bhatia
,
Lone
Baunsgaard
,
Birgitte
Andersen
,
Allan
Svendsen
,
Keith S.
Wilson
Diamond Proposal Number(s):
[7864, 9948]
Open Access
Abstract: Many proteins are synthesized as precursors, with propeptides playing a variety of roles such as assisting in folding or preventing them from being active within the cell. While the precise role of the propeptide in fungal lipases is not completely understood, it was previously reported that mutations in the propeptide region of the Rhizomucor miehei lipase have an influence on the activity of the mature enzyme, stressing the importance of the amino acid composition of this region. We here report two structures of this enzyme in complex with its propeptide, which suggests that the latter plays a role in the correct maturation of the enzyme. Most importantly, we demonstrate that the propeptide shows inhibition of lipase activity in standard lipase assays and propose that an important role of the propeptide is to ensure that the enzyme is not active during its expression pathway in the original host.
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Jun 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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I04-Macromolecular Crystallography
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Pernille
Von Freiesleben
,
Olga V.
Moroz
,
Elena
Blagova
,
Mathias
Wiemann
,
Nikolaj
Spodsberg
,
Jane W.
Agger
,
Gideon J.
Davies
,
Keith S.
Wilson
,
Henrik
Stålbrand
,
Anne S.
Meyer
,
Kristian B. R. M.
Krogh
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Endo-β(1 → 4)-mannanases (endomannanases) catalyse degradation of β-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the −3 and −2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the −2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation.
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Feb 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864, 9948]
Open Access
Abstract: α-Amylases are glycoside hydrolases that break the α-1,4 bonds in starch and related glycans. The degradation of starch is rendered difficult by the presence of varying degrees of α-1,6 branch points and their possible accommodation within the active centre of α-amylase enzymes. Given the myriad industrial uses for starch and thus also for α-amylase-catalysed starch degradation and modification, there is considerable interest in how different α-amylases might accommodate these branches, thus impacting on the potential processing of highly branched post-hydrolysis remnants (known as limit dextrins) and societal applications. Here, it was sought to probe the branch-point accommodation of the Alicyclobacillus sp. CAZy family GH13 α-amylase AliC, prompted by the observation of a molecule of glucose in a position that may represent a branch point in an acarbose complex solved at 2.1 Å resolution. Limit digest analysis by two-dimensional NMR using both pullulan (a regular linear polysaccharide of α-1,4, α-1,4, α-1,6 repeating trisaccharides) and amylopectin starch showed how the Alicyclobacillus sp. enzyme could accept α-1,6 branches in at least the −2, +1 and +2 subsites, consistent with the three-dimensional structures with glucosyl moieties in the +1 and +2 subsites and the solvent-exposure of the −2 subsite 6-hydroxyl group. Together, the work provides a rare insight into branch-point acceptance in these industrial catalysts.
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Jan 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a Kd of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.
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Aug 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1221]
Abstract: Glucoamylases are one of the most important classes of enzymes in the industrial degradation of starch biomass. They consist of a catalytic domain and a carbohydrate-binding domain (CBM), with the latter being important for the interaction with the polymeric substrate. Whereas the catalytic mechanisms and structures of the individual domains are well known, the spatial arrangement of the domains with respect to each other and its influence on activity are not fully understood. Here, the structures of three industrially used fungal glucoamylases, two of which are full length, have been crystallized and determined. It is shown for the first time that the relative orientation between the CBM and the catalytic domain is flexible, as they can adopt different orientations independently of ligand binding, suggesting a role as an anchor to increase the contact time and the relative concentration of substrate near the active site. The flexibility in the orientations of the two domains presented a considerable challenge for the crystallization of the enzymes.
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May 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Olga V.
Moroz
,
Pernille Foged
Jensen
,
Sean P
Mcdonald
,
Nicholas
Mcgregor
,
Elena
Blagova
,
Gerard
Comamala
,
Dorotea R.
Segura
,
Lars
Anderson
,
Santhosh M
Vasu
,
Vasudeva P
Rao
,
Lars
Giger
,
Trine Holst
Sørensen
,
Rune Nygaard
Monrad
,
Allan
Svendsen
,
Jens Erik
Nielsen
,
Bernard
Henrissat
,
Gideon
Davies
,
Harry
Brumer
,
Kasper D.
Rand
,
Keith S.
Wilson
Diamond Proposal Number(s):
[7864]
Abstract: The precise catalytic strategies used for the breakdown of the complex bacterial polysaccharide xanthan, an increasingly frequent component of processed human foodstuffs, have remained a mystery. Here we present the characterization of an endo-xanthanase from Paenibacillus sp. 62047. We show that it is a CAZy family 9 glycoside hydrolase (GH9) responsible for the hydrolysis of the xanthan backbone, capable of generating tetrameric xanthan oligosaccharides from polysaccharide lyase family 8 (PL8) xanthan lyase-treated xanthan. 3-D structure determination reveals a complex multi-modular enzyme in which a catalytic (α/α)6 barrel is flanked by an N-terminal "immunoglobulin-like" (Ig-like) domain (frequently found in GH9 enzymes) and by four additional C-terminal all β-sheet domains which have very few homologs in sequence databases and, at least, one of which functions as a new xanthan-binding domain, now termed CBM84. The solution phase conformation and dynamics of the enzyme in the native calcium-bound state and in the absence of calcium were probed experimentally by hydrogen/deuterium exchange mass spectrometry. Measured conformational dynamics were used to guide the protein engineering of enzyme variants with increased stability in the absence of calcium; a property of interest for the potential use of the enzyme in cleaning detergents. The ability of hydrogen/deuterium exchange mass spectrometry to pinpoint dynamic regions of a protein under stress (e.g. removal of calcium ions) makes this technology a strong tool for improving protein catalyst properties by informed engineering.
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May 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Abstract: Bacterial phosphoinositide-specific phospholipases C (PI-PLCs) are the smallest members of the PI-PLC family, which includes much larger mammalian enzymes responsible for signal transduction as well as enzymes from protozoan parasites, yeast and plants. Eukaryotic PI-PLCs have calcium in the active site, but this is absent in the known structures of Gram-positive bacteria, where its role is instead played by arginine. In addition to their use in a number of industrial applications, the bacterial enzymes attract special interest because they can serve as convenient models of the catalytic domains of eukaryotic enzymes for in vitro activity studies. Here, the structure of a PI-PLC from Pseudomonas sp. 62186 is reported, the first from a Gram-negative bacterium and the first of a native bacterial PI-PLC with calcium present in the active site. Solution of the structure posed particular problems owing to the low sequence identity of available homologous structures. Its dependence on calcium for catalysis makes this enzyme a better model for studies of the mammalian PI-PLCs than the previously used calcium-independent bacterial PI-PLCs.
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Jan 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Abstract: Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~ 10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.
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Jan 2017
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