scholarly journals A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases

2021 ◽  
Vol 296 ◽  
pp. 100500
Author(s):  
Cathleen Kmezik ◽  
Scott Mazurkewich ◽  
Tomke Meents ◽  
Lauren Sara McKee ◽  
Alexander Idström ◽  
...  
Keyword(s):  
Polysaccharide Utilization Locus ◽  
Carbohydrate Esterases

scholarly journals Genome and Secretome Analysis of Staphylotrichum longicolleum DSM105789 Cultured on Agro-Residual and Chitinous Biomass

2021 ◽  
Vol 9 (8) ◽  
pp. 1581
Author(s):  
Arslan Ali ◽  
Bernhard Ellinger ◽  
Sophie C. Brandt ◽  
Christian Betzel ◽  
Martin Rühl ◽  
...  
Keyword(s):  
Sugarcane Bagasse ◽  
Chitinase Activity ◽  
Growth Conditions ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Waste Products ◽  
Enzyme Mixture ◽  
Secretome Analysis ◽  
Polysaccharide Lyases ◽  
Carbohydrate Esterases

Staphylotrichum longicolleum FW57 (DSM105789) is a prolific chitinolytic fungus isolated from wood, with a chitinase activity of 0.11 ± 0.01 U/mg. We selected this strain for genome sequencing and annotation, and compiled its growth characteristics on four different chitinous substrates as well as two agro-industrial waste products. We found that the enzymatic mixture secreted by FW57 was not only able to digest pre-treated sugarcane bagasse, but also untreated sugarcane bagasse and maize leaves. The efficiency was comparable to a commercial enzymatic cocktail, highlighting the potential of the S. longicolleum enzyme mixture as an alternative pretreatment method. To further characterize the enzymes, which efficiently digested polymers such as cellulose, hemicellulose, pectin, starch, and lignin, we performed in-depth mass spectrometry-based secretome analysis using tryptic peptides from in-gel and in-solution digestions. Depending on the growth conditions, we were able to detect from 442 to 1092 proteins, which were annotated to identify from 134 to 224 putative carbohydrate-active enzymes (CAZymes) in five different families: glycoside hydrolases, auxiliary activities, carbohydrate esterases, polysaccharide lyases, glycosyl transferases, and proteins containing a carbohydrate-binding module, as well as combinations thereof. The FW57 enzyme mixture could be used to replace commercial enzyme cocktails for the digestion of agro-residual substrates.

scholarly journals Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
A.E. Naas ◽  
A.K. MacKenzie ◽  
B. Dalhus ◽  
V.G.H. Eijsink ◽  
P.B. Pope
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Structural Features ◽  
Structure And Function ◽  
Dimensional Structure ◽  
Active Site Cleft ◽  
Polysaccharide Utilization Locus ◽  
And Function ◽  
Rumen Metagenome

Abstract Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

Μοριακή, δομική και καταλυτική μελέτη καινοτόμων βιοκαταλυτών (ημικυτταρινάσες) που εμπλέκονται στην αποικοδόμηση της φυτικής βιομάζας

2015 ◽  
Author(s):  
Μαρία-Δέσποινα Χαραυγή
Keyword(s):  
Podospora Anserina ◽  
Myceliophthora Thermophila ◽  
Sporotrichum Thermophile ◽  
Carbohydrate Esterases

Η αποικοδόμηση της φυτικής βιομάζας με τη χρήση ημικυτταρινασών παραμένει ένα από τα προαπαιτούμενα για την αποδοτική παραγωγή βιοκαυσίμων δεύτερης γενιάς από ζυμώσιμα σάκχαρα. Οι γλυκουρονικές εστεράσες (Glucuronoyl Esterases, GEs), ανήκουν σε μια πρόσφατα ανακαλυφθείσα οικογένεια υδατανθρακικών εστερασών (Carbohydrate Esterases, CEs), CE-15, που φέρονται να υδρολύουν τον εστερικό δεσμό μεταξύ των πλευρικών ομάδων του 4-O-μεθυλο-D-γλυκουρονικού οξέος της γλυκουρονοξυλάνης και αρωματικών αλκοολών της λιγνίνης. Ο συγκεκριμένος δεσμός θεωρείται ότι εκπροσωπεί ένα από τα δυσκολότερα στάδια που απαντώνται κατά την πλήρη αποδέσμευση της λιγνίνης από τους υδατάνθρακες που συνθέτουν την ημικυτταρίνη. Στην παρούσα διδακτορική διατριβή δόθηκε έμφαση στην εστεράση του γλυκουρονικού Η αποικοδόμηση της φυτικής βιομάζας με τη χρήση ημικυτταρινασών παραμένει ένα από τα προαπαιτούμενα για την αποδοτική παραγωγή βιοκαυσίμων δεύτερης γενιάς από ζυμώσιμα σάκχαρα. Οι γλυκουρονικές εστεράσες (Glucuronoyl Esterases, GEs), ανήκουν σε μια πρόσφατα ανακαλυφθείσα οικογένεια υδατανθρακικών εστερασών (Carbohydrate Esterases, CEs), CE-15, που φέρονται να υδρολύουν τον εστερικό δεσμό μεταξύ των πλευρικών ομάδων του 4-O-μεθυλο-D-γλυκουρονικού οξέος της γλυκουρονοξυλάνης και αρωματικών αλκοολών της λιγνίνης. Ο συγκεκριμένος δεσμός θεωρείται ότι εκπροσωπεί ένα από τα δυσκολότερα στάδια που απαντώνται κατά την πλήρη αποδέσμευση της λιγνίνης από τους υδατάνθρακες που συνθέτουν την ημικυτταρίνη. Στην παρούσα διδακτορική διατριβή δόθηκε έμφαση στην εστεράση του γλυκουρονικού οξέος αλλά και μεταλλάγματός της από το θερμόφιλο μύκητα Myceliophthora thermophila (συνώνυμο Sporotrichum thermophile, StGE2 και S213A StGE2, αντίστοιχα), και στην εστεράση του γλυκουρονικού οξέος από το μεσόφιλο μικροοργανισμό Podospora anserina (PaGE1). Ειδικότερα, πραγματοποιήθηκαν μελέτες τόσο της StGE2 όσο και της PaGE1 ως προς την υδρόλυση εμπορικά διαθέσιμου συνθετικού υποστρώματος. Η πειραματική μεθοδολογία τριών σταδίων που αναπτύχθηκε αποτελεί έναυσμα για την ανακάλυψη και μελέτη νέων GEs. Παράλληλα, εξετάστηκε η StGE2 ως προς το συνθετικό δυναμικό μέσω αντιδράσεων εστεροποίησης και μετεστεροποίησης σε μη συμβατικά συστήματα που περιελάμβανε επίσης την ακινητοποίησή της. Στην ελεύθερη μορφή του το ένζυμο εμφάνισε ενδείξεις σύνθεσης γλυκουρονιδίων. Επιπροσθέτως, διερευνήθηκε η φυσιολογική δράση του ενζύμου μέσω απομόνωσης λιγνινο-υδατανθρακικών συμπλόκων (Lignin-Carbohydrate Complexes, LCCs) ως φυσικών υποστρωμάτων εμπλουτισμένα σε εστερικούς δεσμούς-στόχο για τις GEs. Η καταλυτική ενεργότητα της StGE2 αλλά και των ημικυτταρινασών που εξετάστηκαν, αποτελεί ένα βήμα προς τη διαλεύκανση του φυσικού ρόλου των GEs. Τέλος, έλαβαν χώρα κρυσταλλογραφικές μελέτες ακτίνων Χ με πρωτεϊνικούς στόχους την StGE2 και την S213A StGE2 που οδήγησαν στον προσδιορισμό των τρισδιάστατων δομών τους σε υψηλή ευκρίνεια 1.55 Å και 1.9 Å, αντίστοιχα, ενώ κατέστη επιτυχής ο προσδιορισμός της τρισδιάστατης κρυσταλλικής δομής του συμπλόκου S213A – MeGlcA σε ευκρίνεια 2.35 Å. Οι συγκεκριμένες δομές αποτελούν τις πρώτες δομές θερμοάντοχης GE, GE που φέρει σημειακή μετάλλαξη σε καταλυτικό αμινοξύ ενώ αυτή του συμπλόκου αποτέλεσε την πρώτη δομή GE με ανάλογο υποστρώματος.

scholarly journals Intestinal IgA Regulates Expression of a Fructan Polysaccharide Utilization Locus in Colonizing Gut Commensal Bacteroides thetaiotaomicron

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Payal Joglekar ◽  
Hua Ding ◽  
Pablo Canales-Herrerias ◽  
Pankaj Jay Pasricha ◽  
Justin L. Sonnenburg ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Ex Vivo ◽  
Microbial Colonization ◽  
Bacteroides Thetaiotaomicron ◽  
Content Type ◽  
Microbial Utilization ◽  
Polysaccharide Utilization Locus ◽  
The Impact

ABSTRACT Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro. Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism. IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.

scholarly journals Multimodular fused acetyl–feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Cathleen Kmezik ◽  
Cyrielle Bonzom ◽  
Lisbeth Olsson ◽  
Scott Mazurkewich ◽  
Johan Larsbrink
Keyword(s):  
Esterase Activity ◽  
Glycoside Hydrolase ◽  
Full Length ◽  
Glycoside Hydrolases ◽  
Corn Cob ◽  
Biomass Degradation ◽  
Carbohydrate Esterase ◽  
Catalytic Domains ◽  
Cob Biomass ◽  
Carbohydrate Esterases

Abstract Background Plant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated. Results Two carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns. Conclusion We demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan and xylan-rich biomass. Moreover, we demonstrated for the first time a synergistic effect between the two fused catalytic domains of a multidomain carbohydrate esterase.

scholarly journals Galactomannan Catabolism Conferred by a Polysaccharide Utilization Locus ofBacteroides ovatus

2016 ◽  
Vol 292 (1) ◽  
pp. 229-243 ◽  
Author(s):  
Viktoria Bågenholm ◽  
Sumitha K. Reddy ◽  
Hanene Bouraoui ◽  
Johan Morrill ◽  
Evelina Kulcinskaja ◽  
...  
Keyword(s):  
Polysaccharide Utilization Locus

scholarly journals The structure of PfGH50B, an agarase from the marine bacterium Pseudoalteromonas fuliginea PS47

2020 ◽  
Vol 76 (9) ◽  
pp. 422-427
Author(s):  
Benjamin Pluvinage ◽  
Craig S. Robb ◽  
Roderick Jeffries ◽  
Alisdair B. Boraston
Keyword(s):  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Structural Features ◽  
Degree Of Polymerization ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
X Ray ◽  
Terminal Domain ◽  
Polysaccharide Utilization Locus ◽  
Hydrolase Family

The recently identified marine bacterium Pseudoalteromonas fuliginea sp. PS47 possesses a polysaccharide-utilization locus dedicated to agarose degradation. In particular, it contains a gene (locus tag EU509_06755) encoding a β-agarase that belongs to glycoside hydrolase family 50 (GH50), PfGH50B. The 2.0 Å resolution X-ray crystal structure of PfGH50B reveals a rare complex multidomain fold that was found in two of the three previously determined GH50 structures. The structure comprises an N-terminal domain with a carbohydrate-binding module (CBM)-like fold fused to a C-terminal domain by a rigid linker. The CBM-like domain appears to function by extending the catalytic groove of the enzyme. Furthermore, the PfGH50B structure highlights key structural features in the mobile loops that may function to restrict the degree of polymerization of the neoagaro-oligosaccharide products and the enzyme processivity.

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