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Larsbrink J, Lo Leggio L Ref: Essays Biochem, :, 2023 : PubMed ESTHER: Larsbrink_2023_Essays.Biochem__ PubMedSearch: Larsbrink 2023 Essays.Biochem PubMedID: 36651189 Abstract Glucuronoyl esterases (GEs) are microbial enzymes able to cleave covalent linkages between lignin and carbohydrates in the plant cell wall. GEs are serine hydrolases found in carbohydrate esterase family 15 (CE15), which belongs to the large alpha/beta hydrolase superfamily. GEs have been shown to reduce plant cell wall recalcitrance by hydrolysing the ester bonds found between glucuronic acid moieties on xylan polysaccharides and lignin. In recent years, the exploration of CE15 has broadened significantly and focused more on bacterial enzymes, which are more diverse in terms of sequence and structure to their fungal counterparts. Similar to fungal GEs, the bacterial enzymes are able to improve overall biomass deconstruction but also appear to have less strict substrate preferences for the uronic acid moiety. The structures of bacterial GEs reveal that they often have large inserts close to the active site, with implications for more extensive substrate interactions than the fungal GEs which have more open active sites. In this review, we highlight the recent work on GEs which has predominantly regarded bacterial enzymes, and discuss similarities and differences between bacterial and fungal enzymes in terms of the biochemical properties, diversity in sequence and modularity, and structural variations that have been discovered thus far in CE15. Title: Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans Mazurkewich S, Scholzen KC, Brusch RH, Poulsen JCN, Theibich Y, Huttner S, Olsson L, Larsbrink J, Lo Leggio L Ref: Acta Crystallographica D Struct Biol, :, 2023 : PubMed ESTHER: Mazurkewich_2023_Acta.Crystallogr.D.Struct.Biol__ PubMedSearch: Mazurkewich 2023 Acta.Crystallogr.D.Struct.Biol PubMedID: 37227091 Gene_locus related to this paper: 9pleo-LfCE15C Abstract In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics. Title: Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum Ristinmaa AS, Coleman T, Cesar L, Langborg Weinmann A, Mazurkewich S, Branden G, Hasani M, Larsbrink J Ref: Journal of Biological Chemistry, 298:101758, 2022 : PubMed ESTHER: Ristinmaa_2022_J.Biol.Chem_298_101758 PubMedSearch: Ristinmaa 2022 J.Biol.Chem 298 101758 PubMedID: 35202648 Gene_locus related to this paper: clobu-CbTan2 Abstract Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallate-binding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass. Title: Mechanism and biomass association of glucuronoyl esterase: an alpha/beta hydrolase with potential in biomass conversion Zong Z, Mazurkewich S, Pereira CS, Fu H, Cai W, Shao X, Skaf MS, Larsbrink J, Lo Leggio L Ref: Nat Commun, 13:1449, 2022 : PubMed ESTHER: Zong_2022_Nat.Commun_13_1449 PubMedSearch: Zong 2022 Nat.Commun 13 1449 PubMedID: 35304453 Gene_locus related to this paper: opitp-b1zmf4 Inhibitor(s) related to this paper: alpha-D-glucopyranuronic-acid, 4-O-Methyl-alpha-D-glucuronate Abstract Glucuronoyl esterases (GEs) are alpha/beta serine hydrolases and a relatively new addition in the toolbox to reduce the recalcitrance of lignocellulose, the biggest obstacle in cost-effective utilization of this important renewable resource. While biochemical and structural characterization of GEs have progressed greatly recently, there have yet been no mechanistic studies shedding light onto the rate-limiting steps relevant for biomass conversion. The bacterial GE OtCE15A possesses a classical yet distinctive catalytic machinery, with easily identifiable catalytic Ser/His completed by two acidic residues (Glu and Asp) rather than one as in the classical triad, and an Arg side chain participating in the oxyanion hole. By QM/MM calculations, we identified deacylation as the decisive step in catalysis, and quantified the role of Asp, Glu and Arg, showing the latter to be particularly important. The results agree well with experimental and structural data. We further calculated the free-energy barrier of post-catalysis dissociation from a complex natural substrate, suggesting that in industrial settings non-catalytic processes may constitute the rate-limiting step, and pointing to future directions for enzyme engineering in biomass utilization. Title: A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases Kmezik C, Mazurkewich S, Meents T, McKee LS, Idstrom A, Armeni M, Savolainen O, Branden G, Larsbrink J Ref: Journal of Biological Chemistry, :100500, 2021 : PubMed ESTHER: Kmezik_2021_J.Biol.Chem__100500 PubMedSearch: Kmezik 2021 J.Biol.Chem 100500 PubMedID: 33667545 Gene_locus related to this paper: 9bact-f8x1n1.1, 9bact-f8x1n1.2 Abstract The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but non-carbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 & 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regards to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multi-gene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii. We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs as well as cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment. Title: Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii Kmezik C, Krska D, Mazurkewich S, Larsbrink J Ref: Sci Rep, 11:17662, 2021 : PubMed ESTHER: Kmezik_2021_Sci.Rep_11_17662 PubMedSearch: Kmezik 2021 Sci.Rep 11 17662 PubMedID: 34480044 Gene_locus related to this paper: 9bace-CE15GH8 Abstract Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions-multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs. Title: Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii Krska D, Mazurkewich S, Brown HA, Theibich Y, Poulsen JN, Morris AL, Koropatkin NM, Lo Leggio L, Larsbrink J Ref: Biochemistry, :, 2021 : PubMed ESTHER: Krska_2021_Biochemistry__ PubMedSearch: Krska 2021 Biochemistry PubMedID: 34180241 Gene_locus related to this paper: calki-e4s6e9 Abstract The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature. Title: Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases Novy V, Carneiro LV, Shin JH, Larsbrink J, Olsson L Ref: Journal of Biological Chemistry, :101302, 2021 : PubMed ESTHER: Novy_2021_J.Biol.Chem__101302 PubMedSearch: Novy 2021 J.Biol.Chem 101302 PubMedID: 34653507 Gene_locus related to this paper: 9pezi-s4vch4, aspfu-q4x1n0, crysp-Q874E9, emeni-q5b2c1, fusso-cutas, pyrbr-Q9Y7G8, copci-b9u443, thite-g2rae6, strsw-c9zcr8 Abstract Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops. Title: The coordinated action of glucuronoyl esterase and alpha-glucuronidase promotes the disassembly of lignin-carbohydrate complexes Raji O, Arnling Baath J, Vuong TV, Larsbrink J, Olsson L, Master ER Ref: FEBS Letters, 595:351, 2021 : PubMed ESTHER: Raji_2021_FEBS.Lett_595_351 PubMedSearch: Raji 2021 FEBS.Lett 595 351 PubMedID: 33277689 Gene_locus related to this paper: tertt-c5bn23 Abstract Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin-carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two alpha-glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing alpha-glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble alpha-glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate. Title: Multimodular fused acetyl-feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass Kmezik C, Bonzom C, Olsson L, Mazurkewich S, Larsbrink J Ref: Biotechnol Biofuels, 13:60, 2020 : PubMed ESTHER: Kmezik_2020_Biotechnol.Biofuels_13_60 PubMedSearch: Kmezik 2020 Biotechnol.Biofuels 13 60 PubMedID: 32266006 Gene_locus related to this paper: 9bact-f8x1n1.1, 9bact-f8x1n1.2 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. Title: Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules Krska D, Larsbrink J Ref: Biotechnol Biofuels, 13:68, 2020 : PubMed ESTHER: Krska_2020_Biotechnol.Biofuels_13_68 PubMedSearch: Krska 2020 Biotechnol.Biofuels 13 68 PubMedID: 32308737 Gene_locus related to this paper: calki-e4s6e9 Abstract BACKGROUND: Efficient degradation of lignocellulosic biomass has become a major bottleneck in industrial processes which attempt to use biomass as a carbon source for the production of biofuels and materials. To make the most effective use of the source material, both the hemicellulosic as well as cellulosic parts of the biomass should be targeted, and as such both hemicellulases and cellulases are important enzymes in biorefinery processes. Using thermostable versions of these enzymes can also prove beneficial in biomass degradation, as they can be expected to act faster than mesophilic enzymes and the process can also be improved by lower viscosities at higher temperatures, as well as prevent the introduction of microbial contamination. RESULTS: This study presents the investigation of the thermostable, dual-function xylanase-glucuronoyl esterase enzyme CkXyn10C-GE15A from the hyperthermophilic bacterium Caldicellulosiruptor kristjanssonii. Biochemical characterization of the enzyme was performed, including assays for establishing the melting points for the different protein domains, activity assays for the two catalytic domains, as well as binding assays for the multiple carbohydrate-binding domains present in CkXyn10C-GE15A. Although the enzyme domains are naturally linked together, when added separately to biomass, the expected boosting of the xylanase action was not seen. This lack of intramolecular synergy might suggest, together with previous data, that increased xylose release is not the main beneficial trait given by glucuronoyl esterases. CONCLUSIONS: Due to its thermostability, CkXyn10C-GE15A is a promising candidate for industrial processes, with both catalytic domains exhibiting melting temperatures over 70 degreesC. Of particular interest is the glucuronoyl esterase domain, as it represents the first studied thermostable enzyme displaying this activity. Title: Structure-function analyses reveal that a glucuronoyl esterase from Teredinibacter turnerae interacts with carbohydrates and aromatic compounds Arnling Baath J, Mazurkewich S, Poulsen JN, Olsson L, Lo Leggio L, Larsbrink J Ref: Journal of Biological Chemistry, 294:6635, 2019 : PubMed ESTHER: Arnling Baath_2019_J.Biol.Chem_294_6635 PubMedSearch: Arnling Baath 2019 J.Biol.Chem 294 6635 PubMedID: 30814248 Gene_locus related to this paper: tertt-c5bn23 Abstract Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages found between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these recalcitrant resources. However, details on how GEs interact with their natural substrates are sparse, calling for thorough structure-function studies. Presented here is the structure and biochemical characterization of a GE, TtCE15A, from the bacterium Teredinibacter turnerae, a symbiont of wood-boring shipworms. To gain deeper insight into enzyme-substrate interactions, inhibition studies were performed with both the WT TtCE15A and variants in which we, by using site-directed mutagenesis, substituted residues suggested to have key roles in binding to or interacting with the aromatic and carbohydrate structures of its uronic acid ester substrates. Our results support the hypothesis that two aromatic residues (Phe-174 and Trp-376), conserved in bacterial GEs, interact with aromatic and carbohydrate structures of these substrates in the enzyme active site, respectively. The solved crystal structure of TtCE15A revealed features previously not observed in either fungal or bacterial GEs, with a large inserted N-terminal region neighboring the active site and a differently positioned residue of the catalytic triad. The findings highlight key interactions between GEs and complex lignin-carbohydrate ester substrates and advance our understanding of the substrate specificities of these enzymes in biomass conversion. Title: Structural and biochemical studies of the glucuronoyl esterase OtCE15A illuminate its interaction with lignocellulosic components Mazurkewich S, Poulsen JN, Lo Leggio L, Larsbrink J Ref: Journal of Biological Chemistry, 294:19978, 2019 : PubMed ESTHER: Mazurkewich_2019_J.Biol.Chem_294_19978 PubMedSearch: Mazurkewich 2019 J.Biol.Chem 294 19978 PubMedID: 31740581 Gene_locus related to this paper: opitp-b1zmf4 Inhibitor(s) related to this paper: beta-D-glucopyranuronic-acid, Aldotetrauronic-acid, beta-D-Xylopyranose, 4-O-Methyl-alpha-D-glucuronate, Xylobiose, Benzyl-D-glucuronoate, D-galacturonate, D-glucuronate, alpha-D-glucopyranuronic-acid Abstract Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these chemically recalcitrant materials. However, details on how GEs interact and catalyze degradation of their natural substrates are sparse, calling for thorough enzyme structure-function studies. GEs belong to carbohydrate esterase family 15 (CE15), which is part of the larger alpha/beta hydrolase superfamily. We present here a structural and mechanistic investigation of the bacterial GE OtCE15A. GEs contain a Ser-His-Asp/Glu catalytic triad, but the location of the catalytic acid in GEs is known to be variable, and OtCE15A possesses two putative catalytic acidic residues in its active site. Through site-directed mutagenesis, we demonstrate here that these residues are functionally redundant, possibly indicating the evolutionary route toward new functionalities within the CE15 family. Structures determined with the bound products glucuronate and galacturonate, as well as a covalently bound intermediate, provided insights into the catalytic mechanism of CE15. A structure of OtCE15A with the glucuronoxylooligosaccharide 2(3)-(4-O-methyl-alpha-D-glucuronyl)-xylotriose (XUX) disclosed that the enzyme can indeed interact with polysaccharides from the plant cell wall, and an additional structure with the disaccharide xylobiose revealed an enzyme surface binding site that might indicate a mechanism by which the enzyme recognizes long glucuronoxylan chains. These results indicate that OtCE15A, and likely most CE15 family enzymes, can utilize glucuronoxylooligosaccharide esters and support the proposal that these enzymes are active on lignin-carbohydrate complexes in plant biomass. Title: Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion Arnling Baath J, Mazurkewich S, Knudsen RM, Poulsen JN, Olsson L, Lo Leggio L, Larsbrink J Ref: Biotechnol Biofuels, 11:213, 2018 : PubMed ESTHER: Arnling Baath_2018_Biotechnol.Biofuels_11_213 PubMedSearch: Arnling Baath 2018 Biotechnol.Biofuels 11 213 PubMedID: 30083226 Gene_locus related to this paper: opitp-b1zmf4, solue-q01ym8 Abstract Background: Lignocellulose is highly recalcitrant to enzymatic deconstruction, where the recalcitrance primarily results from chemical linkages between lignin and carbohydrates. Glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15) have been suggested to play key roles in reducing lignocellulose recalcitrance by cleaving covalent ester bonds found between lignin and glucuronoxylan. However, only a limited number of GEs have been biochemically characterized and structurally determined to date, limiting our understanding of these enzymes and their potential exploration. Results: Ten CE15 enzymes from three bacterial species, sharing as little as 20% sequence identity, were characterized on a range of model substrates; two protein structures were solved, and insights into their regulation and biological roles were gained through gene expression analysis and enzymatic assays on complex biomass. Several enzymes with higher catalytic efficiencies on a wider range of model substrates than previously characterized fungal GEs were identified. Similarities and differences regarding substrate specificity between the investigated GEs were observed and putatively linked to their positioning in the CE15 phylogenetic tree. The bacterial GEs were able to utilize substrates lacking 4-OH methyl substitutions, known to be important for fungal enzymes. In addition, certain bacterial GEs were able to efficiently cleave esters of galacturonate, a functionality not previously described within the family. The two solved structures revealed similar overall folds to known structures, but also indicated active site regions allowing for more promiscuous substrate specificities. The gene expression analysis demonstrated that bacterial GE-encoding genes were differentially expressed as response to different carbon sources. Further, improved enzymatic saccharification of milled corn cob by a commercial lignocellulolytic enzyme cocktail when supplemented with GEs showcased their synergistic potential with other enzyme types on native biomass. Conclusions: Bacterial GEs exhibit much larger diversity than fungal counterparts. In this study, we significantly expanded the existing knowledge on CE15 with the in-depth characterization of ten bacterial GEs broadly spanning the phylogenetic tree, and also presented two novel enzyme structures. Variations in transcriptional responses of CE15-encoding genes under different growth conditions suggest nonredundant functions for enzymes found in species with multiple CE15 genes and further illuminate the importance of GEs in native lignin-carbohydrate disassembly. Title: Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea Huttner S, Nguyen TT, Granchi Z, Chin AWT, Ahren D, Larsbrink J, Thanh VN, Olsson L Ref: Biotechnol Biofuels, 10:265, 2017 : PubMed ESTHER: Huttner_2017_Biotechnol.Biofuels_10_265 PubMedSearch: Huttner 2017 Biotechnol.Biofuels 10 265 PubMedID: 29158777 Abstract Background: Genome and transcriptome sequencing has greatly facilitated the understanding of biomass-degrading mechanisms in a number of fungal species. The information obtained enables the investigation and discovery of genes encoding proteins involved in plant cell wall degradation, which are crucial for saccharification of lignocellulosic biomass in second-generation biorefinery applications. The thermophilic fungus Malbranchea cinnamomea is an efficient producer of many industrially relevant enzymes and a detailed analysis of its genomic content will considerably enhance our understanding of its lignocellulolytic system and promote the discovery of novel proteins. Results: The 25-million-base-pair genome of M. cinnamomea FCH 10.5 was sequenced with 225x coverage. A total of 9437 protein-coding genes were predicted and annotated, among which 301 carbohydrate-active enzyme (CAZyme) domains were found. The putative CAZymes of M. cinnamomea cover cellulases, hemicellulases, chitinases and pectinases, equipping the fungus with the ability to grow on a wide variety of biomass types. Upregulation of 438 and 150 genes during growth on wheat bran and xylan, respectively, in comparison to growth on glucose was revealed. Among the most highly upregulated CAZymes on xylan were glycoside hydrolase family GH10 and GH11 xylanases, as well as a putative glucuronoyl esterase and a putative lytic polysaccharide monooxygenase (LPMO). AA9-domain-containing proteins were also found to be upregulated on wheat bran, as well as a putative cutinase and a protein harbouring a CBM9 domain. Several genes encoding secreted proteins of unknown function were also more abundant on wheat bran and xylan than on glucose. Conclusions: The comprehensive combined genome and transcriptome analysis of M. cinnamomea provides a detailed insight into its response to growth on different types of biomass. In addition, the study facilitates the further exploration and exploitation of the repertoire of industrially relevant lignocellulolytic enzymes of this fungus. | |||||||
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