Publications by authors named "Finn L Aachmann"

66 Publications

The impact of reductants on the catalytic efficiency of a lytic polysaccharide monooxygenase and the special role of dehydroascorbic acid.

FEBS Lett 2021 Nov 29. Epub 2021 Nov 29.

Faculty of Chemistry, Biotechnology and Food Science, NMBU - Norwegian University of Life Sciences, 1432, Ås, Norway.

Monocopper lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of glycosidic bonds in a reductant-dependent reaction. Recent studies indicate that LPMOs, rather than being O -dependent monooxygenases, are H O -dependent peroxygenases. Here, we describe SscLPMO10B, a novel LPMO from the phytopathogenic bacterium Streptomyces scabies and address links between this enzyme's catalytic rate and in situ hydrogen peroxide production in the presence of ascorbic acid, gallic acid and l-cysteine. Studies of Avicel degradation showed a clear correlation between the catalytic rate of SscLPMO10B and the rate of H O generation in the reaction mixture. We also assessed the impact of oxidized ascorbic acid, dehydroascorbic acid (DHA), on LPMO activity, since DHA, which is not considered a reductant, was recently reported to drive LPMO reactions. Kinetic studies, combined with NMR analysis, showed that DHA is unstable and converts into multiple derivatives, some of which are redox active and can fuel the LPMO reaction by reducing the active site copper and promoting H O production. These results show that the apparent monooxygenase activity observed in SscLPMO10B reactions without exogenously added H O reflects a peroxygenase reaction.
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http://dx.doi.org/10.1002/1873-3468.14246DOI Listing
November 2021

Structural and functional variation of chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus.

J Biol Chem 2021 10 17;297(4):101084. Epub 2021 Aug 17.

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Trondheim, Norway. Electronic address:

Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. However, structural and functional properties of some CBMs remain unknown, and it is not clear why some LPMOs, like CjLPMO10A from the soil bacterium Cellvibrio japonicus, have multiple CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on their functional variation and determined the solution structures of both by NMR, which constitutes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with K values in the micromolar range, CjCBM73 has higher affinity for chitin than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding of CjCBM73 to this chitooligosaccharide was detected. These functional differences correlate with distinctly different arrangements of three conserved aromatic amino acids involved in substrate binding. In CjCBM5, these residues show a linear arrangement that seems compatible with the experimentally observed affinity for single chitin chains. On the other hand, the arrangement of these residues in CjCBM73 suggests a wider binding surface that may interact with several chitin chains. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.
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http://dx.doi.org/10.1016/j.jbc.2021.101084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8449059PMC
October 2021

Structure-function analysis of a new PL17 oligoalginate lyase from the marine bacterium Zobellia galactanivorans DsijT.

Glycobiology 2021 Nov;31(10):1364-1377

Sorbonne Université, CNRS, Integrative Biology of Marine Models (LBI2M), Station Biologique de Roscoff (SBR), 29680 Roscoff, France.

Alginate is a major compound of brown macroalgae and as such an important carbon and energy source for heterotrophic marine bacteria. Despite the rather simple composition of alginate only comprising mannuronate and guluronate units, these bacteria feature complex alginolytic systems that can contain up to seven alginate lyases. This reflects the necessity of large enzyme systems for the complete degradation of the abundant substrate. Numerous alginate lyases have been characterized. They belong to different polysaccharide lyase (PL) families, but only one crystal structure of a family 17 (PL17) alginate lyase has been reported to date, namely Alg17c from the gammaproteobacterium Saccharophagus degradans. Biochemical and structural characterizations are helpful to link sequence profiles to function, evolution of functions and niche-specific characteristics. Here, we combined detailed biochemical and crystallographic analysis of AlyA3, a PL17 alginate lyase from the marine flavobacteria Zobellia galactanivorans DsijT, providing the first structure of a PL17 in the Bacteroidetes phylum. AlyA3 is exo-lytic and highly specific of mannuronate stretches. As part of an "alginate utilizing locus", its activity is complementary to that of other characterized alginate lyases from the same bacterium. Structural comparison with Alg17c highlights a common mode of action for exo-lytic cleavage of the substrate, strengthening our understanding of the PL17 catalytic mechanism. We show that unlike Alg17c, AlyA3 contains an inserted flexible loop at the entrance to the catalytic groove, likely involved in substrate recognition, processivity and turn over.
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http://dx.doi.org/10.1093/glycob/cwab058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8600288PMC
November 2021

RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork.

Nucleic Acids Res 2021 04;49(7):3948-3966

Department of Clinical and Molecular Medicine, NTNU Norwegian University of Science and Technology, NO-7491 Trondheim, Norway.

Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2-3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential 'pre-replicative' removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.
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http://dx.doi.org/10.1093/nar/gkab195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053108PMC
April 2021

Impact of Alginate Mannuronic-Guluronic Acid Contents and pH on Protein Binding Capacity and Complex Size.

Biomacromolecules 2021 02 8;22(2):649-660. Epub 2021 Jan 8.

Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby Denmark.

Alginates, serving as hydrocolloids in the food and pharma industries, form particles at pH < 4.5 with positively charged proteins, such as β-lactoglobulin (β-Lg). Alginates are linear anionic polysaccharides composed of 1,4-linked β-d-mannuronate (M) and α-l-guluronate (G) residues. The impact of M and G contents and pH is investigated to correlate with the formation and size of β-Lg alginate complexes under relevant ionic strength. It is concluded, using three alginates of M/G ratios 0.6, 1.1, and 1.8 and similar molecular mass, that β-Lg binding capacity is higher at pH 4.0 than at pH 2.65 and for high M content. By contrast, the largest particles are obtained at pH 2.65 and with high G content. At pH 4.0 and 2.65, the stoichiometry was 28-48 and 3-10 β-Lg molecules bound per alginate, respectively, increasing with higher M content. The findings will contribute to the design of formation of the desired alginate-protein particles in the acidic pH range.
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http://dx.doi.org/10.1021/acs.biomac.0c01485DOI Listing
February 2021

Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase.

Appl Environ Microbiol 2021 02 26;87(6). Epub 2021 Feb 26.

Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly-β-d-mannuronate, and poly-α-l-guluronate within a broad range of pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l--5-hexulosuronate), which are formed after ring opening, and, finally, two epimers of a 5-member hemiketal called 4-deoxy-d--hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (NMR) assignment of these hemiketals refine our current understanding of alginate degradation. The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases, and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here, we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.
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http://dx.doi.org/10.1128/AEM.02399-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105002PMC
February 2021

Class IV Lasso Peptides Synergistically Induce Proliferation of Cancer Cells and Sensitize Them to Doxorubicin.

iScience 2020 Dec 10;23(12):101785. Epub 2020 Nov 10.

Department of Pharmacognosy, University of Vienna, Vienna 1090, Austria.

Heterologous expression of a biosynthesis gene cluster from sp. resulted in the discovery of two unique class IV lasso peptides, felipeptins A1 and A2. A mixture of felipeptins stimulated proliferation of cancer cells, while having no such effect on the normal cells. Detailed investigation revealed, that pre-treatment of cancer cells with a mixture of felipeptins resulted in downregulation of the tumor suppressor Rb, making the cancer cells to proliferate faster. Pre-treatment with felipeptins made cancer cells considerably more sensitive to the anticancer agent doxorubicin and re-sensitized doxorubicin-resistant cells to this drug. Structural characterization and binding experiments showed an interaction between felipeptins resulting in complex formation, which explains their synergistic effect. This discovery may open an alternative avenue in cancer treatment, helping to eliminate quiescent cells that often lead to cancer relapse.
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http://dx.doi.org/10.1016/j.isci.2020.101785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689547PMC
December 2020

H, C, N resonance assignment of the apo form of the small, chitin-active lytic polysaccharide monooxygenase JdLPMO10A from Jonesia denitrificans.

Biomol NMR Assign 2021 04 19;15(1):79-84. Epub 2020 Nov 19.

NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491, Trondheim, Norway.

The lytic polysaccharide monooxygenase JdLPMO10A is the N-terminal domain of the multimodular protein Jd1381. The isolated JdLPMO10A domain is one of the smallest chitin-active lytic polysaccharide monooxygenases known to date with a size of only 15.5 kDa. JdLPMO10A is a copper-dependent oxidative enzyme that depolymerizes chitin by hydroxylating the C1 carbon in the glycosidic bond. JdLPMO10A has been isotopically labeled and recombinantly expressed. Here, we report the H, C, N resonance assignment of JdLPMO10A. Secondary structural elements predicted based on the NMR assignment are in excellent agreement with the crystal structure of JdLPMO10A.
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http://dx.doi.org/10.1007/s12104-020-09986-zDOI Listing
April 2021

Bi-Functional Alginate Oligosaccharide-Polymyxin Conjugates for Improved Treatment of Multidrug-Resistant Gram-Negative Bacterial Infections.

Pharmaceutics 2020 Nov 11;12(11). Epub 2020 Nov 11.

Advanced Therapies Group, School of Dentistry, College of Biomedical and Life Sciences, Cardiff University, Heath Park, Cardiff CF14 4XY, UK.

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide ("OligoG")-polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG-polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200-12,800 g/mol and antibiotic loading of 6.1-12.9% /, were reproducibly synthesised. In vitro inflammatory cytokine production (tumour necrosis factor alpha (TNFα) ELISA) and cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of colistin (2.2-9.3-fold) and polymyxin B (2.9-27.2-fold) were significantly decreased by OligoG conjugation. Antimicrobial susceptibility tests (minimum inhibitory concentration (MIC), growth curves) demonstrated similar antimicrobial efficacy of ester- and amide-linked conjugates to that of the parent antibiotic but with more sustained inhibition of bacterial growth. OligoG-polymyxin conjugates exhibited improved selectivity for Gram-negative bacteria in comparison to mammalian cells (approximately 2-4-fold). Both OligoG-colistin conjugates caused significant disruption of biofilm formation and induced bacterial death (confocal laser scanning microscopy). When conjugates were tested in an in vitro "time-to-kill" (TTK) model using , only ester-linked conjugates reduced viable bacterial counts (~2-fold) after 4 h. Bi-functional OligoG-polymyxin conjugates have potential therapeutic benefits in the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, directly reducing toxicity whilst retaining antimicrobial and antibiofilm activities.
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http://dx.doi.org/10.3390/pharmaceutics12111080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696216PMC
November 2020

Synthesis of glycoconjugates utilizing the regioselectivity of a lytic polysaccharide monooxygenase.

Sci Rep 2020 08 6;10(1):13197. Epub 2020 Aug 6.

Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Chr. M. Falsens vei 1, Ås, Norway.

Polysaccharides from plant biomass are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. Potential applications of glycoconjugates include therapeutics and drug delivery, vaccine development and as fine chemicals. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifications, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful enzymes known for their application in cellulose degradation, specifically oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specific approach to produce oxime-based glycoconjugates from LPMO-functionalized oligosaccharides. The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes.
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http://dx.doi.org/10.1038/s41598-020-69951-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411024PMC
August 2020

Mechanistic basis of substrate-O coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study.

Proc Natl Acad Sci U S A 2020 08 28;117(32):19178-19189. Epub 2020 Jul 28.

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology and Food Science, Norwegian University of Science and Technology, N-7491 Trondheim, Norway;

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of -LPMO10A from , along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and N-HYSCORE spectroscopy study on the uniformly isotope-labeled Cu(II)-bound N-LPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x-y) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O activation in chitin-active AA10 LPMOs.
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http://dx.doi.org/10.1073/pnas.2004277117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431007PMC
August 2020

Functional characterization of three Azotobacter chroococcum alginate-modifying enzymes related to the Azotobacter vinelandii AlgE mannuronan C-5-epimerase family.

Sci Rep 2020 07 27;10(1):12470. Epub 2020 Jul 27.

Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælandsvei 6/8, 7491, Trondheim, Norway.

Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.
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http://dx.doi.org/10.1038/s41598-020-68789-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385640PMC
July 2020

2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides.

Biomacromolecules 2020 07 26;21(7):2884-2895. Epub 2020 Jun 26.

NOBIPOL, Department of Biotechnology and Food Science, NTNU - Norwegian University of Science and Technology, Sem Saelands veg 6/8, NO-7491 Trondheim, Norway.

Diblock oligosaccharides based on renewable resources allow for a range of new but, so far, little explored biomaterials. Coupling of blocks through their reducing ends ensures retention of many of their intrinsic properties that otherwise are perturbed in classical lateral modifications. Chitin is an abundant, biodegradable, bioactive, and self-assembling polysaccharide. However, most coupling protocols relevant for chitin blocks have shortcomings. Here we exploit the highly reactive 2,5-anhydro-d-mannose residue at the reducing end of chitin oligomers obtained by nitrous acid depolymerization. Subsequent activation by dihydrazides or dioxyamines provides precursors for chitin-based diblock oligosaccharides. These reactions are much faster than for other carbohydrates, and only acyclic imines (hydrazones or oximes) are formed (no cyclic -glycosides). α-Picoline borane and cyanoborohydride are effective reductants of imines, but in contrast to most other carbohydrates, they are not selective for the imines in the present case. This could be circumvented by a simple two-step procedure. Attachment of a second block to hydrazide- or aminooxy-functionalized chitin oligomers turned out to be even faster than the attachment of the first block. The study provides simple protocols for the preparation of chitin--chitin and chitin--dextran diblock oligosaccharides without involving protection/deprotection strategies.
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http://dx.doi.org/10.1021/acs.biomac.0c00620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660591PMC
July 2020

Identification of a Pivotal Residue for Determining the Block Structure-Forming Properties of Alginate C-5 Epimerases.

ACS Omega 2020 Mar 24;5(8):4352-4361. Epub 2020 Feb 24.

Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Norwegian Biopolymer Laboratory (NOBIPOL), Sem Sælands vei 6/8, NO 7491 Trondheim, Norway.

Alginate is a linear copolymer composed of 1→4 linked β-d-mannuronic acid (M) and its epimer α-l-guluronic acid (G). The polysaccharide is first produced as homopolymeric mannuronan and subsequently, at the polymer level, C-5 epimerases convert M residues to G residues. The bacterium encodes a family of seven secreted and calcium ion-dependent mannuronan C-5 epimerases (AlgE1-AlgE7). These epimerases consist of two types of structural modules: the A-modules, which contain the catalytic site, and the R-modules, which influence activity through substrate and calcium binding. In this study, we rationally designed new hybrid mannuronan C-5 epimerases constituting the A-module from AlgE6 and the R-module from AlgE4. This led to a better understanding of the molecular mechanism determining differences in MG- and GG-block-forming properties of the enzymes. A long loop with either tyrosine or phenylalanine extruding from the β-helix of the enzyme proved essential in defining the final alginate block structure, probably by affecting substrate binding. Normal mode analysis of the A-module from AlgE6 supports the results.
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http://dx.doi.org/10.1021/acsomega.9b04490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057702PMC
March 2020

Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides.

Carbohydr Polym 2020 Mar 23;232:115748. Epub 2019 Dec 23.

NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, NO-7491 Trondheim, Norway. Electronic address:

Reducing end activation of poly- and oligosaccharides by bifunctional dioxyamines and dihydrazides enables aniline-free and cyanoborohydride-free conjugation to aldehyde-containing molecules, particles and surfaces without compromising the chain structure. Chitosans are due to their polycationic character, biodegradability, and bioactivity important candidates for conjugation. Here, we present a kinetic and structural study of the conjugation of a dioxyamine and a dihydrazide to enzymatically produced chitooligosaccharides ranging from N,N'-diacetylchitobiose to a decamer, all having N-acetyl d-glucosamine at the reducing end. Conjugation of the dioxyamine resulted in mixtures of (E)- and (Z)-oximes and β-N-pyranoside, whereas the dihydrazide yielded cyclic N-glycosides. Reaction kinetics was essentially independent of DP. Stable secondary amines were in both cases obtained by reduction with α-picoline borane, but higher temperatures were needed to obtain acceptable reduction rate. Comparison to dextran oligomers shows that the nature of the reducing end strongly influences the kinetics of both the conjugation and reduction.
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http://dx.doi.org/10.1016/j.carbpol.2019.115748DOI Listing
March 2020

NMR and Fluorescence Spectroscopies Reveal the Preorganized Binding Site in Family 14 Carbohydrate-Binding Module from Human Chitotriosidase.

ACS Omega 2019 Dec 9;4(26):21975-21984. Epub 2019 Dec 9.

Department of Biotechnology and Food Science, Norwegian Biopolymer Laboratory (NOBIPOL), NTNU Norwegian University of Science and Technology, Trondheim 7491, Norway.

Carbohydrate-binding modules (CBM) play important roles in targeting and increasing the concentration of carbohydrate active enzymes on their substrates. Using NMR to get the solution structure of CBM14, we can gain insight into secondary structure elements and intramolecular interactions with our assigned nuclear overhauser effect peaks. This reveals that two conserved aromatic residues (Phe437 and Phe456) make up the hydrophobic core of the CBM. These residues are also responsible for connecting the two β-sheets together, by being part of β2 and β4, respectively, and together with disulfide bridges, they create CBM14's characteristic "hevein-like" fold. Most CBMs rely on aromatic residues for substrate binding; however, CBM14 contains just a single tryptophan (Trp465) that together with Asn466 enables substrate binding. Interestingly, an alanine mutation of a single residue (Leu454) located behind Trp465 renders the CBM incapable of binding. Fluorescence spectroscopy performed on this mutant reveals a significant blue shift, as well as a minor blue shift for its neighbor Val455. The reduction in steric hindrance causes the tryptophan to be buried into the hydrophobic core of the structure and therefore suggests a preorganized binding site for this CBM. Our results show that both Trp465 and Asn466 are affected when CBM14 interacts with both (GlcNAc) and β-chitin, that the binding interactions are weak, and that CBM14 displays a slightly higher affinity toward β-chitin.
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http://dx.doi.org/10.1021/acsomega.9b03043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933781PMC
December 2019

Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe .

J Biol Chem 2019 11 17;294(47):17915-17930. Epub 2019 Sep 17.

Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium (PL6). The structure of recombinant PL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of PL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for attack correlated with M specificity. Using biochemical analyses, we found that PL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, PL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased PL6 activity on alginate 7-fold. LC-electrospray ionization-MS quantification of products and lack of activity on NaBH-reduced octa-mannuronic acid indicated that PL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.
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http://dx.doi.org/10.1074/jbc.RA119.010206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879350PMC
November 2019

Efficient Grafting of Cyclodextrin to Alginate and Performance of the Hydrogel for Release of Model Drug.

Sci Rep 2019 06 27;9(1):9325. Epub 2019 Jun 27.

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology and Food Science, NTNU - Norwegian University of Science and Technology, N-7491, Trondheim, Norway.

Controlling the rate of release of molecules from a hydrogel is of high interest for various drug delivery systems and medical devices. A strategy to alter the release profiles of soluble and poorly soluble active ingredients from hydrogels can be to combine the hydrogel forming ability of alginate with the inclusion forming ability of cyclodextrins (CyD). Here, β-CyD was grafted to alginate in a three-step synthesis using periodate oxidation, reductive amination and copper(I)-catalyzed azide-alkyne cycloaddition. A grafting degree of 4.7% mol β-CyD/mol sugar residues was obtained. The grafting degree was controlled by varying the reaction parameters where the amount of linker used in reductive amination was especially influential. Ca-alginate gel beads grafted with β-CyD showed increased uptake of the model molecule methyl orange. Release experiments showed that the grafted material had a prolonged release of methyl orange and an increased total amount of released methyl orange. These results show that the β-CyD grafted alginate is still able to form a hydrogel while the grafted cyclodextrins retain their ability to form inclusion complex with methyl orange. Further testing should be done with this system to investigate capability for drug delivery applications.
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http://dx.doi.org/10.1038/s41598-019-45761-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597533PMC
June 2019

Chitin-Active Lytic Polysaccharide Monooxygenases.

Adv Exp Med Biol 2019 ;1142:115-129

Department of Biotechnology and Food Science, NOBIPOL, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, N-7491, Trondheim, Norway.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the cleavage of 1,4-glycosidic bonds various plant cell wall polysaccharides and chitin. In contrast to glycoside hydrolases, LPMOs are active on the crystalline regions of polysaccharides and thus synergize with hydrolytic enzymes. This synergism leads to an overall increase in the biomass-degradation activity of enzyme mixtures. Chitin-active LPMOs were discovered in 2010 and are currently classified in families AA10, AA11, and AA15 of the Carbohydrate-Active enZYmes database, which include LPMOs from bacteria, fungi, insects, and viruses. LPMOs have become important enzymes both industrially and scientifically and, in this chapter, we provide a brief introduction to chitin-active LPMOs including a summary of the 20+ chitin-active LPMOs that have been characterized so far. Then, we describe their structural features, catalytic mechanism, and appended carbohydrate modules. Finally, we show how chitin-active LPMOs can be used to perform chemo-enzymatic modification of chitin substrates.
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http://dx.doi.org/10.1007/978-981-13-7318-3_6DOI Listing
August 2019

Mechanical Properties of Ca-Saturated Hydrogels with Functionalized Alginate.

Gels 2019 Apr 19;5(2). Epub 2019 Apr 19.

NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

In this work, the mechanical properties and stability of alginate hydrogels containing functionalized alginates (peptide and β-cyclodextrin) were studied. There is an increasing interest in the modification of alginates to add functions such as cell attachment and increased solubility of hydrophobic drugs, for better performance in tissue engineering and drug release, respectively. Functionalization was achieved in this study via periodate oxidation followed by reductive amination, previously shown to give a high and controllable degree of substitution. Young's modulus and the stress at rupture of the hydrogels were in general lowered when exchanging native alginate with the modified alginate. Still, the gel strength could be adjusted by the fraction of modified alginate in the mixed hydrogels as well as the degree of oxidation. No notable difference in deformation at rupture was observed while syneresis was influenced by the degree of oxidation and possibly by the nature and amount of the grafted molecules. The mixed hydrogels were less stable than hydrogels with only native alginate, and modified alginate was released from the hydrogels. Furthermore, the hydrogels in general rather disintegrated than swelled upon saline treatments.
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http://dx.doi.org/10.3390/gels5020023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631140PMC
April 2019

Polysaccharide degradation by lytic polysaccharide monooxygenases.

Curr Opin Struct Biol 2019 12 1;59:54-64. Epub 2019 Apr 1.

Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), N-1432 Ås, Norway. Electronic address:

The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has had a major impact on our current understanding of the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. The number of LPMO sequence families keeps expanding and novel substrate specificities and biological functionalities are being discovered. The catalytic mechanism of these LPMOs remains somewhat enigmatic. Recently, novel insights have been obtained from studies of enzyme-substrate complexes by X-ray crystallography, EPR, NMR, and modeling. Furthermore, it has been shown that LPMOs may carry out peroxygenase reactions, at much higher rates than monooxygenase reactions, which affects our understanding and exploitation of these powerful enzymes.
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http://dx.doi.org/10.1016/j.sbi.2019.02.015DOI Listing
December 2019

Production, Characterization, and Application of an Alginate Lyase, AMOR_PL7A, from Hot Vents in the Arctic Mid-Ocean Ridge.

J Agric Food Chem 2019 Mar 25;67(10):2936-2945. Epub 2019 Feb 25.

Faculty of Chemistry, Biotechnology and Food Science , Norwegian University of Life Sciences (NMBU) , P.O. Box 5003, N-1432 Aas , Norway.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. We describe a thermostable alginate lyase belonging to Polysaccharide Lyase family 7 (PL7), which can be used to degrade brown seaweed, Saccharina latissima, at conditions also suitable for a commercial cellulase cocktail (Cellic CTec2). This enzyme, AMOR_PL7A, is a β-d-mannuronate specific (EC 4.2.2.3) endoacting alginate lyase, which degrades alginate and poly mannuronate within a broad range of pH, temperature and salinity. At 65 °C and pH 6.0, its Km and k values for sodium alginate are 0.51 ± 0.09 mg/mL and 7.8 ± 0.3 s respectively. Degradation of seaweed with blends of Cellic CTec2 and AMOR_PL7A at 55 °C in seawater showed that the lyase efficiently reduces viscosity and increases glucose solublization. Thus, AMOR_PL7A may be useful in development of efficient protocols for enzymatic seaweed processing.
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http://dx.doi.org/10.1021/acs.jafc.8b07190DOI Listing
March 2019

The edible mushroom Albatrellus ovinus contains a α-l-fuco-α-d-galactan, α-d-glucan, a branched (1 → 6)-β-d-glucan and a branched (1 → 3)-β-d-glucan.

Carbohydr Res 2019 Jan 1;471:28-38. Epub 2018 Nov 1.

Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, NOBIPOL, NO-7491, Trondheim, Norway.

Albatrellus ovinus, the sheep polypore, is a large, dense mushroom being rich in cell wall material. Polysaccharides were isolated by sequential extraction, enzymatic treatment and analyzed with respect to monosaccharide composition, glycosidic linkages by methylation and GC-MS as well as NMR spectroscopy. A fucogalactan composed of an (1 → 6)-α-d-galactan backbone with single α-l-Fucp residues attached at O-2 was identified in the hot water extract obtained after treatment with a protease and size exclusion chromatography. Both the hot water extract and the hot alkali extract contained an (1 → 4)-α-d-glucan whereas β-d-glucans were mainly present in the latter. Structural analysis suggested the presence of two different β-d-glucan backbone structures; a (1 → 6)-linked β-d-glucan with single β-d-Glcp residues at O-3 and also a (1 → 3)-linked β-d-glucan with branches in O-6. In addition there were identified short (1 → 2)-linked β-d-xylan and (1 → 3)-α-d-mannan chains.
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http://dx.doi.org/10.1016/j.carres.2018.10.012DOI Listing
January 2019

Resonance assignments for the apo-form of the cellulose-active lytic polysaccharide monooxygenase TaLPMO9A.

Biomol NMR Assign 2018 10 16;12(2):357-361. Epub 2018 Aug 16.

NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491, Trondheim, Norway.

The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the H, C, and N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts.
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http://dx.doi.org/10.1007/s12104-018-9839-yDOI Listing
October 2018

Mode of action and specificity of a chitinase from unicellular microalgae, Euglena gracilis.

Plant Mol Biol 2018 Aug 6;97(6):553-564. Epub 2018 Aug 6.

Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara, 631-8505, Japan.

Key Message: Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites - 2 and - 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase. The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (A, n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites - 2 and - 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites - 2 and - 1.
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http://dx.doi.org/10.1007/s11103-018-0759-0DOI Listing
August 2018

A colorimetric assay to rapidly determine the activities of lytic polysaccharide monooxygenases.

Biotechnol Biofuels 2018 2;11:215. Epub 2018 Aug 2.

1Division of Glycoscience, Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Center, 106 91 Stockholm, Sweden.

Background: Lytic polysaccharide monooxygenase (LPMOs) are enzymes that catalyze the breakdown of polysaccharides in biomass and have excellent potential for biorefinery applications. However, their activities are relatively low, and methods to measure these activities are costly, tedious or often reflect only an apparent activity to the polysaccharide substrates. Here, we describe a new method we have developed that is simple to use to determine the activities of type-1 (C1-oxidizing) LPMOs. The method is based on quantifying the ionic binding of cations to carboxyl groups formed by the action of type-1 LPMOs on polysaccharides. It allows comparisons to be made of activities under different conditions.

Results: Based on the colorimetric detection and quantification of the pyrocatechol violet (PV)-Ni complex, we have developed an assay to reliably detect and quantify carboxylate moieties introduced by type-1 LPMOs. Conditions were optimized for determining the activities of specific LPMOs. Comparisons were made of the activities against cellulose and chitin of a novel AA10 LPMO and a recently reported family AA11 LPMO. Activities of both LPMOs were boosted by hydrogen peroxide in the 1st hour of the reaction, with a 16-fold increase for the family AA11 LPMO, and up to a 34-fold increase for the family AA10 LPMO.

Conclusions: We developed a versatile colorimetric cation-based assay to determine the activities of type-1 LPMOs. The assay is quick, low cost and could be adapted for use in industrial biorefineries.
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http://dx.doi.org/10.1186/s13068-018-1211-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071379PMC
August 2018

Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation.

Protein Sci 2018 09;27(9):1636-1650

Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.

The catalytically crucial N-terminal histidine (His1) of fungal lytic polysaccharide monooxygenases (LPMOs) is post-translationally modified to carry a methylation. The functional role of this methylation remains unknown. We have carried out an in-depth functional comparison of two variants of a family AA9 LPMO from Thermoascus aurantiacus (TaLPMO9A), one with, and one without the methylation on His1. Various activity assays showed that the two enzyme variants are identical in terms of substrate preferences, cleavage specificities and the ability to activate molecular oxygen. During the course of this work, new functional features of TaLPMO9A were discovered, in particular the ability to cleave xyloglucan, and these features were identical for both variants. Using a variety of techniques, we further found that methylation has minimal effects on the pK of His1, the affinity for copper and the redox potential of bound copper. The two LPMOs did, however, show clear differences in their resistance against oxidative damage. Studies with added hydrogen peroxide confirmed recent claims that low concentrations of H O boost LPMO activity, whereas excess H O leads to LPMO inactivation. The methylated variant of TaLPMO9A, produced in Aspergillus oryzae, was more resistant to excess H O and showed better process performance when using conditions that promote generation of reactive-oxygen species. LPMOs need to protect themselves from reactive oxygen species generated in their active sites and this study shows that methylation of the fully conserved N-terminal histidine provides such protection.
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http://dx.doi.org/10.1002/pro.3451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194291PMC
September 2018

The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation.

J Biol Chem 2018 08 2;293(34):13006-13015. Epub 2018 Jul 2.

From NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, N-7491 Trondheim, Norway,

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of polysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes. The catalytic domains of a considerable fraction of LPMOs and other carbohydrate-active enzymes (CAZymes) are tethered to carbohydrate-binding modules (CBMs) by flexible linkers. These linkers preclude X-ray crystallographic studies, and the functional implications of these modular assemblies remain partly unknown. Here, we used NMR spectroscopy to characterize structural and dynamic features of full-length modular LPMO10C from We observed that the linker is disordered and extended, creating distance between the CBM and the catalytic domain and allowing these domains to move independently of each other. Functional studies with cellulose nanofibrils revealed that most of the substrate-binding affinity of full-length LPMO10C resides in the CBM. Comparison of the catalytic performance of full-length LPMO10C and its isolated catalytic domain revealed that the CBM is beneficial for LPMO activity at lower substrate concentrations and promotes localized and repeated oxidation of the substrate. Taken together, these results provide a mechanistic basis for understanding the interplay between catalytic domains linked to CBMs in LPMOs and CAZymes in general.
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http://dx.doi.org/10.1074/jbc.RA118.004269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109919PMC
August 2018

Analytical Tools for Characterizing Cellulose-Active Lytic Polysaccharide Monooxygenases (LPMOs).

Methods Mol Biol 2018 ;1796:219-246

Biotechnology and Food Science, Faculty of Chemistry, Norwegian University of Life Sciences, Ås, Norway.

Lytic polysaccharide monooxygenases are copper-dependent enzymes that perform oxidative cleavage of glycosidic bonds in cellulose and various other polysaccharides. LPMOs acting on cellulose use a reactive oxygen species to abstract a hydrogen from the C1 or C4, followed by hydroxylation of the resulting substrate radical. The resulting hydroxylated species is unstable, resulting in glycoside bond scission and formation of an oxidized new chain end. These oxidized chain ends are spontaneously hydrated at neutral pH, leading to formation of an aldonic acid or a gemdiol, respectively. LPMO activity may be characterized using a variety of analytic tools, the most common of which are high-performance anion exchange chromatography system with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry (MALDI-MS). NMR may be used to increase the certainty of product identifications, in particular the site of oxidation. Kinetic studies of LPMOs have several pitfalls and to avoid these, it is important to secure copper saturation, avoid the presence of free transition metals in solution, and control the amount of reductant (i.e., electron supply to the LPMO). Further insight into LPMO properties may be obtained by determining the redox potential and by determining the affinity for copper. In some cases, substrate affinity can be assessed using isothermal titration calorimetry. These methods are described in this chapter.
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http://dx.doi.org/10.1007/978-1-4939-7877-9_16DOI Listing
February 2019

Preparation of 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid (DEH) and Guluronic Acid Rich Alginate Using a Unique exo-Alginate Lyase from Thalassotalea crassostreae.

J Agric Food Chem 2018 Feb 6;66(6):1435-1443. Epub 2018 Feb 6.

Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center , Stockholm, SE-106 91, Sweden.

Marine multicellular algae are considered promising crops for the production of sustainable biofuels and commodity chemicals. However, their commercial exploitation is currently limited by a lack of appropriate and efficient enzymes for converting alginate into metabolizable building blocks, such as 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Herein, we report the discovery and characterization of a unique exo-alginate lyase from the marine bacterium Thalassotalea crassostreae that possesses excellent catalytic efficiency against poly-β-D-mannuronate (poly M) alginate, with a k of 135.8 s, and a 5-fold lower k of 25 s against poly-α-L-guluronate (poly G alginate). We propose that this preference for poly M is due to a structural feature of the protein's active site. The mode of action and specificity of this enzyme has made it possible to design an effective and environmentally friendly process for the production of DEH and low molecular weight guluronate-enriched alginate.
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http://dx.doi.org/10.1021/acs.jafc.7b05751DOI Listing
February 2018
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