I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Olga
Moroz
,
Elena
Blagova
,
Andrey A.
Lebedev
,
Lars K.
Skov
,
Roland A.
Pache
,
Kirk M.
Schnorr
,
Lars
Kiemer
,
Esben P.
Friis
,
Søren
Nymand-Grarup
,
Li
Ming
,
Liu
Ye
,
Mikkel
Klausen
,
Marianne T.
Cohn
,
Esben G. W.
Schmidt
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[7864, 13587, 24948]
Open Access
Abstract: Muramidases (also known as lysozymes) hydrolyse the peptidoglycan component of the bacterial cell wall and are found in many glycoside hydrolase (GH) families. Similar to other glycoside hydrolases, muramidases sometimes have noncatalytic domains that facilitate their interaction with the substrate. Here, the identification, characterization and X-ray structure of a novel fungal GH24 muramidase from Trichophaea saccata is first described, in which an SH3-like cell-wall-binding domain (CWBD) was identified by structure comparison in addition to its catalytic domain. Further, a complex between a triglycine peptide and the CWBD from T. saccata is presented that shows a possible anchor point of the peptidoglycan on the CWBD. A `domain-walking' approach, searching for other sequences with a domain of unknown function appended to the CWBD, was then used to identify a group of fungal muramidases that also contain homologous SH3-like cell-wall-binding modules, the catalytic domains of which define a new GH family. The properties of some representative members of this family are described as well as X-ray structures of the independent catalytic and SH3-like domains of the Kionochaeta sp., Thermothielavioides terrestris and Penicillium virgatum enzymes. This work confirms the power of the module-walking approach, extends the library of known GH families and adds a new noncatalytic module to the muramidase arsenal.
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Aug 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598, 24948]
Open Access
Abstract: Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.
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Aug 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 α-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.
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May 2023
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I04-Macromolecular Crystallography
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Olga V.
Moroz
,
Elena
Blagova
,
Andrey A.
Lebedev
,
Filomeno
Sanchez Rodriguez
,
Daniel J.
Rigden
,
Jeppe
Wegener Tams
,
Reinhard
Wilting
,
Jan Kjølhede
Vester
,
Emily
Longhi
,
Gustav
Hammerich Hansen
,
Kristian
Bertel Rømer Mørkeberg Krogh
,
Roland A.
Pache
,
Gideon
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[18598]
Abstract: β-Galactosidases catalyse the hydrolysis of lactose into galactose and glucose; as an alternative reaction, some β-galactosidases also catalyse the formation of galactooligosaccharides by transglycosylation. Both reactions have industrial importance: lactose hydrolysis is used to produce lactose-free milk, while galactooligosaccharides have been shown to act as prebiotics. For some multi-domain β-galactosidases, the hydrolysis/transglycosylation ratio can be modified by the truncation of carbohydrate-binding modules. Here, an analysis of BbgIII, a multidomain β-galactosidase from Bifidobacterium bifidum, is presented. The X-ray structure has been determined of an intact protein corresponding to a gene construct of eight domains. The use of evolutionary covariance-based predictions made sequence docking in low-resolution areas of the model spectacularly easy, confirming the relevance of this rapidly developing deep-learning-based technique for model building. The structure revealed two alternative orientations of the CBM32 carbohydrate-binding module relative to the GH2 catalytic domain in the six crystallographically independent chains. In one orientation the CBM32 domain covers the entrance to the active site of the enzyme, while in the other orientation the active site is open, suggesting a possible mechanism for switching between the two activities of the enzyme, namely lactose hydrolysis and transgalactosylation. The location of the carbohydrate-binding site of the CBM32 domain on the opposite site of the module to where it comes into contact with the catalytic GH2 domain is consistent with its involvement in adherence to host cells. The role of the CBM32 domain in switching between hydrolysis and transglycosylation modes offers protein-engineering opportunities for selective β-galactosidase modification for industrial purposes in the future.
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Dec 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Olga V.
Moroz
,
Elena
Blagova
,
Edward
Taylor
,
Johan
Turkenburg
,
Lars K.
Skov
,
Garry P.
Gippert
,
Kirk M.
Schnorr
,
Li
Ming
,
Liu
Ye
,
Mikkel
Klausen
,
Marianne T.
Cohn
,
Esben G. W.
Schmidt
,
Søren
Nymand-Grarup
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[13587, 7864]
Open Access
Abstract: Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities, cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.
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Mar 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: α-mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297, and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer associated N-glycosylation.
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Jul 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587, 18598]
Abstract: Alternatives to petroleum-based chemicals are highly sought-after for on-going efforts to reduce the damaging effects of human activity on the environment. Copper radical oxidases from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts because they oxidize primary alcohols in a chemo-selective manner without complex organic cofactors. However, despite numerous studies on canonical galactose oxidases (GalOx, EC 1.1.3.9) and engineered variants, and the recent discovery of a Colletotrichum graminicola copper radical alcohol oxidase (AlcOx, EC 1.1.3.13), the catalytic potentials of very few AA5_2 members have been characterized. Guided by sequence similarity network and phylogenetic analyses, in this study we targeted a distinct paralog from the fungus C. graminicola as a representative member of a large uncharacterized subgroup of AA5_2. Through recombinant production and detailed kinetic analysis, we demonstrated that this enzyme is weakly active towards carbohydrates, but efficiently catalyzes the oxidation of aryl alcohols to the corresponding aldehydes. As such, this represents the initial characterization of a demonstrable aryl alcohol oxidase (AAO, EC 1.1.3.7) in AA5, an activity which is classically associated with flavin-dependent glucose-methanol-choline (GMC) oxidoreductases of Auxiliary Activity Family 3 (AA3). X-ray crystallography revealed a distinct multidomain architecture comprising an N-terminal PAN domain abutting a canonical AA5 seven-bladed propeller catalytic domain. Of direct relevance to biomass processing, the wild-type enzyme exhibits the highest activity on the primary alcohol of 5-hydroxymethylfurfural (HMF), a product of significant interest in the lignocellulosic bio-refinery concept. Thus, the chemoselective oxidation of HMF to 2,5-diformylfuran (DFF) by C. graminicola aryl alcohol oxidase (CgrAAO) from AA5 provides a fundamental building block for chemistry via biotechnology.
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Feb 2020
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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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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
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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