Mazurkewich S

References (12)

Title : Exploration of three Dyadobacter fermentans enzymes uncovers molecular activity determinants in CE15 - Carbonaro_2024_Appl.Microbiol.Biotechnol_108_335
Author(s) : Carbonaro M , Mazurkewich S , Fiorentino G , Lo Leggio L , Larsbrink J
Ref : Applied Microbiology & Biotechnology , 108 :335 , 2024
Abstract : Glucuronoyl esterases (GEs) are serine-type hydrolase enzymes belonging to carbohydrate esterase family 15 (CE15), and they play a central role in the reduction of recalcitrance in plant cell walls by cleaving ester linkages between glucuronoxylan and lignin in lignocellulose. Recent studies have suggested that bacterial CE15 enzymes are more heterogeneous in terms of sequence, structure, and substrate preferences than their fungal counterparts. However, the sequence space of bacterial GEs has still not been fully explored, and further studies on diverse enzymes could provide novel insights into new catalysts of biotechnological interest. To expand our knowledge on this family of enzymes, we investigated three unique CE15 members encoded by Dyadobacter fermentans NS114(T), a Gram-negative bacterium found endophytically in maize/corn (Zea mays). The enzymes are dissimilar, sharing >= 39% sequence identity to each other' and were considerably different in their activities towards synthetic substrates. Combined analysis of their primary sequences and structural predictions aided in establishing hypotheses regarding specificity determinants within CE15, and these were tested using enzyme variants attempting to shift the activity profiles. Together, the results expand our existing knowledge of CE15, shed light into the molecular determinants defining specificity, and support the recent thesis that diverse GEs encoded by a single microorganism may have evolved to fulfil different physiological functions. KEY POINTS: D. fermentans encodes three CE15 enzymes with diverse sequences and specificities The Region 2 inserts in bacterial GEs may directly influence enzyme activity Rational amino acid substitutions improved the poor activity of the DfCE15A enzyme.
ESTHER : Carbonaro_2024_Appl.Microbiol.Biotechnol_108_335
PubMedSearch : Carbonaro_2024_Appl.Microbiol.Biotechnol_108_335
PubMedID: 38747981

Title : Polysaccharide utilization loci from Bacteroidota encode CE15 enzymes with possible roles in cleaving pectin-lignin bonds - Seveso_2024_Appl.Environ.Microbiol__e0176823
Author(s) : Seveso A , Mazurkewich S , Banerjee S , Poulsen JC , Lo Leggio L , Larsbrink J
Ref : Applied Environmental Microbiology , :e0176823 , 2024
Abstract : The plant cell wall is a highly complex matrix, and while most of its polymers interact non-covalently, there are also covalent bonds between lignin and carbohydrates. Bonds between xylan and lignin are known, such as the glucuronoyl ester bonds that are cleavable by CE15 enzymes. Our work here indicates that enzymes from CE15 may also have other activities, as we have discovered enzymes in PULs proposed to target other polysaccharides, including pectin. Our study represents the first investigation of such enzymes. Our first hypothesis that the enzymes would act as pectin methylesterases was shown to be false, and we instead propose that they may cleave other esters on complex pectins such as rhamnogalacturonan II. The work presents both the characterization of five novel enzymes and can also provide indirect information about the components of the cell wall itself, which is a highly challenging material to chemically analyze in fine detail.
ESTHER : Seveso_2024_Appl.Environ.Microbiol__e0176823
PubMedSearch : Seveso_2024_Appl.Environ.Microbiol__e0176823
PubMedID: 38179933
Gene_locus related to this paper: phov8-a6kwt9

Title : Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans - Mazurkewich_2023_Acta.Crystallogr.D.Struct.Biol__
Author(s) : 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
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.
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

Title : Mechanism and biomass association of glucuronoyl esterase: an alpha\/beta hydrolase with potential in biomass conversion - Zong_2022_Nat.Commun_13_1449
Author(s) : 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
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.
ESTHER : Zong_2022_Nat.Commun_13_1449
PubMedSearch : Zong_2022_Nat.Commun_13_1449
PubMedID: 35304453
Gene_locus related to this paper: opitp-b1zmf4

Title : Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum - Ristinmaa_2022_J.Biol.Chem_298_101758
Author(s) : 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
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, 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.
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

Title : A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases - Kmezik_2021_J.Biol.Chem__100500
Author(s) : 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
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.
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

Title : Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii - Kmezik_2021_Sci.Rep_11_17662
Author(s) : Kmezik C , Krska D , Mazurkewich S , Larsbrink J
Ref : Sci Rep , 11 :17662 , 2021
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.
ESTHER : Kmezik_2021_Sci.Rep_11_17662
PubMedSearch : Kmezik_2021_Sci.Rep_11_17662
PubMedID: 34480044
Gene_locus related to this paper: 9bace-CE15GH8

Title : Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii - Krska_2021_Biochemistry__
Author(s) : Krska D , Mazurkewich S , Brown HA , Theibich Y , Poulsen JN , Morris AL , Koropatkin NM , Lo Leggio L , Larsbrink J
Ref : Biochemistry , : , 2021
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.
ESTHER : Krska_2021_Biochemistry__
PubMedSearch : Krska_2021_Biochemistry__
PubMedID: 34180241
Gene_locus related to this paper: calki-e4s6e9

Title : Multimodular fused acetyl-feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass - Kmezik_2020_Biotechnol.Biofuels_13_60
Author(s) : Kmezik C , Bonzom C , Olsson L , Mazurkewich S , Larsbrink J
Ref : Biotechnol Biofuels , 13 :60 , 2020
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.
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

Title : Structural and biochemical studies of the glucuronoyl esterase OtCE15A illuminate its interaction with lignocellulosic components - Mazurkewich_2019_J.Biol.Chem_294_19978
Author(s) : Mazurkewich S , Poulsen JN , Lo Leggio L , Larsbrink J
Ref : Journal of Biological Chemistry , 294 :19978 , 2019
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.
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

Title : Structure-function analyses reveal that a glucuronoyl esterase from Teredinibacter turnerae interacts with carbohydrates and aromatic compounds - Arnling Baath_2019_J.Biol.Chem_294_6635
Author(s) : Arnling Baath J , Mazurkewich S , Poulsen JN , Olsson L , Lo Leggio L , Larsbrink J
Ref : Journal of Biological Chemistry , 294 :6635 , 2019
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.
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

Title : Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion - Arnling Baath_2018_Biotechnol.Biofuels_11_213
Author(s) : Arnling Baath J , Mazurkewich S , Knudsen RM , Poulsen JN , Olsson L , Lo Leggio L , Larsbrink J
Ref : Biotechnol Biofuels , 11 :213 , 2018
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.
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