I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Abstract: Pectins are a major dietary nutrient source for the human gut microbiota (HGM). The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases (PMEs) essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II-derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric and nuclear magnetic resonance approaches revealed that it is a bi-methylated nona-oligosaccharide GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an alpha/beta (α/β) hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for PMEs, which are predominantly right-handed β-helical proteins. Bioinformatics analyses revealed that the family is dominated by sequences from the prominent genera of the HGM, including Bacteroides and Prevotella. Our results not only highlight the critical role played by this family of enzymes in pectin metabolism but provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.
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Oct 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Cecilia
Piergentili
,
Jennifer
Ross
,
Didi
He
,
Kelly J.
Gallagher
,
Will A.
Stanley
,
Laurène
Adam
,
C. Logan
Mackay
,
Arnaud
Basle
,
Kevin J.
Waldron
,
David J.
Clarke
,
Jon
Marles-wright
Diamond Proposal Number(s):
[9487]
Abstract: Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and neighboring secondary metal binding sites. We have previously identified a putative metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.
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Sep 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Lucy I.
Crouch
,
Marcelo V.
Liberato
,
Paulina A.
Urbanowicz
,
Arnaud
Basle
,
Christopher A.
Lamb
,
Christopher J.
Stewart
,
Katie
Cooke
,
Mary
Doona
,
Stephanie
Needham
,
Richard R.
Brady
,
Janet E.
Berrington
,
Katarina
Madunic
,
Manfred
Wuhrer
,
Peter
Chater
,
Jeffery P.
Pearson
,
Robert
Glowacki
,
Eric C.
Martens
,
Fuming
Zhang
,
Robert J.
Linhardt
,
Daniel I. R.
Spencer
,
David N.
Bolam
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.
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Aug 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Anna
Barwinska-sendra
,
Yuritzi M.
Garcia
,
Kacper M.
Sendra
,
Arnaud
Basle
,
Eilidh S.
Mackenzie
,
Emma
Tarrant
,
Patrick
Card
,
Leandro C.
Tabares
,
Cédric
Bicep
,
Sun
Un
,
Thomas E.
Kehl-fie
,
Kevin J.
Waldron
Diamond Proposal Number(s):
[7864, 9948, 18598]
Open Access
Abstract: Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal’s redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.
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Jun 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Mariusz
Madej
,
Joshua B. R.
White
,
Zuzanna
Nowakowska
,
Shaun
Rawson
,
Carsten
Scavenius
,
Jan J.
Enghild
,
Grzegorz P.
Bereta
,
Karunakar
Pothula
,
Ulrich
Kleinekathoefer
,
Arnaud
Basle
,
Neil A.
Ranson
,
Jan
Potempa
,
Bert
Van Den Berg
Diamond Proposal Number(s):
[13587]
Abstract: Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a ‘pedal bin’ mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.
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May 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Many biologists are now routinely seeking to determine the three-dimensional structures of their proteins of choice, illustrating the importance of this knowledge, but also of the simplification and streamlining of structure-determination processes. Despite the fact that most software packages offer simple pipelines, for the non-expert navigating the outputs and understanding the key aspects can be daunting. Here, the structure determination of the type IV pili (TFP) protein PilA1 from Clostridioides difficile is used to illustrate the different steps involved, the key decision criteria and important considerations when using the most common pipelines and software. Molecular-replacement pipelines within CCP4i2 are presented to illustrate the more commonly used processes. Previous knowledge of the biology and structure of TFP pilins, particularly the presence of a long, N-terminal α-helix required for pilus formation, allowed informed decisions to be made during the structure-determination strategy. The PilA1 structure was finally successfully determined using ARCIMBOLDO and the ab initio MR strategy used is described.
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Mar 2020
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Open Access
Abstract: This study describes a method to estimate the likelihood of success in determining a macromolecular structure by X-ray crystallography and experimental single-wavelength anomalous dispersion (SAD) or multiple-wavelength anomalous dispersion (MAD) phasing based on initial data-processing statistics and sample crystal properties. Such a predictive tool can rapidly assess the usefulness of data and guide the collection of an optimal data set. The increase in data rates from modern macromolecular crystallography beamlines, together with a demand from users for real-time feedback, has led to pressure on computational resources and a need for smarter data handling. Statistical and machine-learning methods have been applied to construct a classifier that displays 95% accuracy for training and testing data sets compiled from 440 solved structures. Applying this classifier to new data achieved 79% accuracy. These scores already provide clear guidance as to the effective use of computing resources and offer a starting point for a personalized data-collection assistant.
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Mar 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the ‘sulfation problem’ is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.
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Jan 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Justina
Briliūtė
,
Paulina A.
Urbanowicz
,
Ana S.
Luis
,
Arnaud
Basle
,
Neil
Paterson
,
Osmond
Rebello
,
Jenifer
Hendel
,
Didier A.
Ndeh
,
Elisabeth C.
Lowe
,
Eric C.
Martens
,
Daniel I. R.
Spencer
,
David N.
Bolam
,
Lucy I.
Crouch
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.
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Jun 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Abstract: The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGIIdegrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the D-galacturonic acid-α-1,2-L-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second D-galacturonic acid side chain (linked β-1,3 to L-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double-displacement, retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/β)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting D-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes.
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Mar 2019
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