Lo Leggio L

References (9)

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 : Glucuronoyl esterases - enzymes to decouple lignin and carbohydrates and enable better utilization of renewable plant biomass - Larsbrink_2023_Essays.Biochem__
Author(s) : Larsbrink J , Lo Leggio L
Ref : Essays Biochem , : , 2023
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.
ESTHER : Larsbrink_2023_Essays.Biochem__
PubMedSearch : Larsbrink_2023_Essays.Biochem__
PubMedID: 36651189

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 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 : 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