| 1. |
- Anderson, Lars, et al.
(författare)
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Kinetics and stereochemistry of the Cellulomonas fimi beta-mannanase studied using H-1-NMR
- 2008
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 26:1-2, s. 86-95
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Tidskriftsartikel (refereegranskat)abstract
- Endo-1,4-beta-mannanases (beta-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The beta-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 beta-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by H-1-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10(-5) s(-1) at 5 degrees C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the -3 subsite had a greater impact on the apparent k(cat)/K-m ratio (the catalytic efficiency) than the +2 subsite. The beta-anomer of mannotriose was hydrolysed 1000-times more efficiently than the alpha-anomer indicating selectivity for the beta- over the alpha-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.
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| 2. |
- Arnling Bååth, Jenny, 1987, et al.
(författare)
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Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
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Tidskriftsartikel (refereegranskat)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.
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| 3. |
- Arnling Bååth, Jenny, 1987, et al.
(författare)
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Structure-function analyses reveal that a glucuronoyl esterase from Teredinibacter turnerae interacts with carbohydrates and aromatic compounds
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:16, s. 6635-6644
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Tidskriftsartikel (refereegranskat)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 structurefunction 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.
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| 4. |
- Brander, Søren, et al.
(författare)
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Biochemical evidence of both copper chelation and oxygenase activity at the histidine brace
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.
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| 5. |
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| 6. |
- Hekmat, Omid, et al.
(författare)
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Rational Engineering of Mannosyl Binding in the Distal Glycone Subsites of Cellulomonas fimi Endo-beta-1,4-mannanase: Mannosyl Binding Promoted at Subsite-2 and Demoted at Subsite-3
- 2010
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:23, s. 4884-4896
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Tidskriftsartikel (refereegranskat)abstract
- To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1, 4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce.japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C.fimi doubly substituted mannanase was determined to 2.35 angstrom resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of O-18-labeled products obtained using (H2O)-O-18 as a solvent. The results obtained with oligosacharide substrates show that although the catalytic efficiency (k(cat)/K-m) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K-m decreased similar to 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.
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| 7. |
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| 8. |
- Mazurkewich, Scott, 1982, et al.
(författare)
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Biochemical and structural investigation of the CE15 family: Glucuronoyl esterases acting on recalcitrant biomass
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Glucuronoyl esterases (GEs) are a relatively new class of enzymes which cleave ester linkages between lignin and glucuronoxylan. GEs have been identified in many biomass-degrading microbes and are now classified into the Carbohydrate Esterase Family 15 (CE15). The CE15 family is diverse (as low as 20% sequence identity), however to-date only a few GEs from a small clade of the CE15 phylogenetic tree have been biochemically characterized and only two protein structures have been solved. To investigate the diversity of CE15 members, we have studied a broad range of proteins from across the phylogenetic tree. Enzymes were biochemically characterized and three-dimensional structures for two of the enzymes were solved. Analysis of the structures suggest possible binding sites for lignin fragments and xylooligosaccharides that have not been previously reported. Investigations into the molecular determinants supporting the potential binding sites is being pursued.
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| 9. |
- Mazurkewich, Scott, 1982, et al.
(författare)
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Mechanism of the glucuronoyl esterase OtCE15A: an alpha/beta hydrolase involved with biomass conversion
- 2022
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Glucuronoyl esterases are α/β serine hydrolases from Carbohydrate Esterase family 15 (CE15) which cleave an ester linkage that connects lignin to glucuronyl xylan, an important linkage contributing to biomass recalcitrance. Presented here is our recent work on probing the enzyme-substrate interactions of a model glucuronoyl esterase, Ot CE15A from the soil bacterium Opitutus terrae , through combined structural, kinetic, and QM/MM investigations. These new investigations greatly add to the knowledge of enzyme-substrate interactions in CE15 and enhances our understanding as to how these enzymes may act in natural conditions, which could aid in industrial biomass conversion technologies.
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| 10. |
- Mazurkewich, Scott, 1982, et al.
(författare)
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Structural and biochemical studies of the glucuronoyl esterase OtCE15A illuminate its interaction with lignocellulosic components
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:52, s. 19978-19987
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Tidskriftsartikel (refereegranskat)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 α/β hydrolase superfamily. We present here a structural and mechanistic investigation of the bacterial GE Ot CE15A. GEs contain a Ser–His–Asp/Glu catalytic triad, but the location of the catalytic acid in GEs is known to be variable, and Ot CE15A 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 Ot CE15A with the glucuronoxylooligosaccharide 23-(4- O -methyl-α-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 Ot CE15A, 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.
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