| 1. |
- Frandsen, K. E. H., et al.
(author)
-
The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases
- 2016
-
In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 12:4, s. 298-
-
Journal article (peer-reviewed)abstract
- Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.
|
|
| 2. |
- Krska, Daniel, 1989, et al.
(author)
-
Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii
- 2021
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 60:27, s. 2206-2220
-
Journal article (peer-reviewed)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.
|
|
| 3. |
- Carbonaro, Miriam, et al.
(author)
-
Exploration of three Dyadobacter fermentans enzymes uncovers molecular activity determinants in CE15
- 2024
-
In: Applied Microbiology and Biotechnology. - 1432-0614 .- 0175-7598. ; 108:1
-
Journal article (peer-reviewed)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 NS114T, 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.
|
|
| 4. |
- Larsbrink, Johan, 1982, et al.
(author)
-
Glucuronoyl esterases - enzymes to decouple lignin and carbohydrates and enable better utilization of renewable plant biomass
- 2023
-
In: Essays in biochemistry. - 0071-1365. ; 67:3, s. 493-503
-
Research review (peer-reviewed)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 α/β 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.
|
|
| 5. |
- Le Nours, K, et al.
(author)
-
The structure and characterization of a modular endo-beta-1,4-mannanase from Cellulomonas fimi
- 2005
-
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:38, s. 12700-12708
-
Journal article (peer-reviewed)abstract
- The endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi is a modular plant cell wall degrading enzyme involved in the hydrolysis of the backbone of mannan, one of the most abundant polysaccharides of the hemicellulosic network in the plant cell wall. The crystal structure of a recombinant truncated endo-beta-1,4-mannanase from C. fimi (CMan26A-50K) was determined by X-ray crystallography to 2.25 angstrom resolution using the molecular replacement technique. The overall structure of the enzyme consists of a core (beta/alpha)(8)-barrel catalytic module characteristic of clan GH-A, connected via a linker to an immunoglobulin-like module of unknown function. A complex with the oligosaccharide mannotriose to 2.9 angstrom resolution has also been obtained. Both the native structure and the complex show a cacodylate ion bound at the -1 subsite, while subsites -2, -3, and -4 are occupied by mannotriose in the complex. Enzyme kinetic analysis and the analysis of hydrolysis products from manno-oligosaccharides and mannopentitol suggest five important active-site cleft subsites. CfMan26A-50K has a high affinity -3 subsite with Phe325 as an aromatic platform, which explains the mannose releasing property of the enzyme. Structural differences with the homologous Cellvibrio japonicus beta-1,4-mannanase (CjMan26A) at the -2 and -3 subsites may explain the poor performance of CfMan26A mutants as "glycosynthases".
|
|
| 6. |
|
|
| 7. |
- Sprenger, Janina, et al.
(author)
-
Crystal structures of Val58Ile tryptophan repressor in a domain-swapped array in the presence and absence of l-tryptophan Sprenger Janina
- 2021
-
In: Acta Crystallographica Section F: Structural Biology Communications. - 2053-230X. ; 77, s. 215-225
-
Journal article (peer-reviewed)abstract
- The crystal structures of domain-swapped tryptophan repressor (TrpR) variant Val58Ile before and after soaking with the physiological ligand l-tryptophan (l-Trp) indicate that l-Trp occupies the same location in the domain-swapped form as in native dimeric TrpR and makes equivalent residue contacts. This result is unexpected because the ligand binding-site residues arise from three separate polypeptide chains in the domain-swapped form. This work represents the first published structure of a domain-swapped form of TrpR with l-Trp bound. The presented structures also show that the protein amino-terminus, whether or not it bears a disordered extension of about 20 residues, is accessible in the large solvent channels of the domain-swapped crystal form, as in the structures reported previously in this form for TrpR without N-terminal extensions. These findings inspire the exploration of l-Trp analogs and N-terminal modifications as labels to orient guest proteins that cannot otherwise be crystallized in the solvent channels of crystalline domain-swapped TrpR hosts for potential diffraction analysis.
|
|
| 8. |
- Sprenger, Janina, et al.
(author)
-
Guest-protein incorporation into solvent channels of a protein host crystal (hostal)
- 2021
-
In: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 471-485
-
Journal article (peer-reviewed)abstract
- Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals (here given the name hostals) incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ∼2.5 Å can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required.
|
|
