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  • 1. Becker, D.
    et al.
    Braet, C.
    Brumer, Harry
    KTH, Superseded Departments, Biotechnology.
    Claeyssens, M.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Fagerstrom, B. R.
    Harris, M.
    Jones, T. A.
    Kleywegt, G. J.
    Koivula, A.
    Mahdi, S.
    Piens, K.
    Sinnott, M. L.
    Stahlberg, J.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Underwood, M.
    Wohlfahrt, G.
    Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant2001In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 356, p. 19-30Article in journal (Refereed)
    Abstract [en]

    The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

  • 2.
    Divne, Christina
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    SINNING, I
    STAHLBERG, J
    PETTERSSON, G
    BAILEY, M
    SIIKAAHO, M
    MARGOLLESCLARK, E
    TEERI, T
    JONES, TA
    CRYSTALLIZATION AND PRELIMINARY-X-RAY STUDIES ON THE CORE PROTEINS OF CELLOBIOHYDROLASE-I AND ENDOGLUCANASE-I FROM TRICHODERMA-REESEI1993In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 234, no 3, p. 905-907Article in journal (Refereed)
  • 3.
    DIVNE, Christina
    et al.
    KTH, School of Biotechnology (BIO).
    STAHLBERG, J
    REINIKAINEN, T
    RUOHONEN, L
    PETTERSSON, G
    KNOWLES, JKC
    TEERI, TT
    JONES, TA
    THE 3-DIMENSIONAL CRYSTAL-STRUCTURE OF THE CATALYTIC CORE OF CELLOBIOHYDROLASE-I FROM TRICHODERMA-REESEI1994In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 265, no 5171, p. 524-528Article in journal (Refereed)
  • 4.
    Divne, Christina
    et al.
    KTH, School of Biotechnology (BIO).
    Stahlberg, J
    Teeri, T T
    Jones, T A
    High-resolution crystal structures reveal how a cellulose chain is bound in the 50 angstrom long tunnel of cellobiohydrolase I from Trichoderma reesei1998In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 275, no 2, p. 309-325Article in journal (Refereed)
  • 5.
    Eklöf, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO).
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 82009In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, no 4, p. 1029-1036Article in journal (Refereed)
  • 6.
    Gandini, Rosaria
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Reichenbach, Tom
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Structural basis for dolichylphosphate mannose biosynthesis2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, no 1, article id 120Article in journal (Refereed)
    Abstract [en]

    Protein glycosylation is a critical protein modification. In biogenic membranes of eukaryotes and archaea, these reactions require activated mannose in the form of the lipid conjugate dolichylphosphate mannose (Dol-P-Man). The membrane protein dolichylphosphate mannose synthase (DPMS) catalyzes the reaction whereby mannose is transferred from GDP-mannose to the dolichol carrier Dol-P, to yield Dol-P-Man. Failure to produce or utilize Dol-P-Man compromises organism viability, and in humans, several mutations in the human dpm1 gene lead to congenital disorders of glycosylation (CDG). Here, we report three high-resolution crystal structures of archaeal DPMS from Pyrococcus furiosus, in complex with nucleotide, donor, and glycolipid product. The structures offer snapshots along the catalytic cycle, and reveal how lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol-P-Man synthesis. The structures also rationalize the loss of dolichylphosphate mannose synthase function in dpm1-associated CDG.The generation of glycolipid dolichylphosphate mannose (Dol-P-Man) is a critical step for protein glycosylation and GPI anchor synthesis. Here the authors report the structure of dolichylphosphate mannose synthase in complex with bound nucleotide and donor to provide insight into the mechanism of Dol-P-Man synthesis.

  • 7.
    Gullfot, Fredrika
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    von Schantz, Laura
    Karlsson, Eva Nordberg
    Ohlin, Mats
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module2010In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 78, no 3, p. 785-789Article in journal (Refereed)
  • 8. Hallberg, B. M.
    et al.
    Bergfors, T.
    Backbro, K.
    Pettersson, G.
    Henriksson, Gunnar
    KTH, Superseded Departments, Pulp and Paper Technology.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    A new scaffold for binding haem in the cytochrome domain of the extracellular flavocytochrome cellobiose dehydrogenase2000In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 8, no 1, p. 79-88Article in journal (Refereed)
    Abstract [en]

    Background: The fungal oxidoreductase cellobiose dehydrogenase (CDH) degrades both lignin and cellulose, and is the only known extracellular flavocytochrome. This haemoflavoenzyme has a multidomain organisation with a b-type cytochrome domain linked to a large flavodehydrogenase domain. The two domains can be separated proteolytically to yield a functional cytochrome and a flavodehydrogenase. Here, we report the crystal structure of the cytochrome domain of CDH. Results: The crystal structure of the b-type cytochrome domain of CDH from the wood-degrading fungus Phanerochaete chrysosporium has been determined at 1.9 Angstrom resolution using multiple isomorphous replacement ncluding anomalous scattering information. Three models of the cytochrome have been refined: the in vitro prepared cytochrome in its redox-inactive state (pH 7.5) and redox-active state (pH 4.6), as well as the naturally occurring cytochrome fragment. Conclusions: The 190-residue long cytochrome domain of CDH folds as a beta sandwich with the topology of the antibody Fab V-H domain. The haem iron is ligated by Met65 and His 163, which confirms previous results from spectroscopic studies. This is only the second example of a b-type cytochrome with this ligation, the first being cytochrome b(562). The haem-propionate groups are surface exposed and, therefore, might play a role in the association between the cytochrome and flavoprotein domain, and in interdomain electron transfer. There are no large differences in overall structure of the cytochrome at redoxactive pH as compared with the inactive form, which excludes the possibility that pH-dependent redox inactivation results from partial denaturation. From the electron-density map of the naturally occurring cytochrome, we conclude that it corresponds to the proteolytically prepared cytochrome domain.

  • 9. Hallberg, B. M.
    et al.
    Henriksson, Gunnar
    KTH, Superseded Departments, Pulp and Paper Technology.
    Pettersson, G.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Crystal structure of the flavoprotein domain of the extracellular flavocytochrome cellobiose dehydrogenase2002In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 315, no 3, p. 421-434Article in journal (Refereed)
    Abstract [en]

    Cellobiose dehydrogenase (CDH) participates in the degradation of cellulose and lignin. The protein is an extracellular flavocytochrome with a b-type cytochrome domain (CYTcdh) connected to a flavodehydrogenase domain (DHcdh). DHcdh catalyses a two-electron oxidation at the anomeric C1 position of cellobiose to yield cellobiono-1,5-lactone, and the electrons are subsequently transferred from DHcdh to an acceptor, either directly or via CYTcdh. Here, we decribe the crystal structure of Phanerochaete chrysosporium DHcdh determined at 1.5 Angstrom resolution. DHcdh belongs to the GMC family of oxidoreductases, which includes glucose oxidase (GOX) and cholesterol oxidase (COX); however, the sequence identity with members of the family is low. The overall fold of DHcdh is p-hydroxybenzoate hydroxylase-like and is similar to, but also different from, that of GOX and COX. It is partitioned into an FAD-binding subdomain of alpha/beta type and a substrate-binding subdomain consisting of a seven-stranded beta sheet and six helices. Docking of CYTcdh, and DHcdh suggests that CYTcdh covers the active-site entrance in DHcdh, and that the resulting distance between the cofactors is within acceptable limits for inter-domain electron transfer. Based on docking of the substrate, cellobiose, in the active site of DHcdh, we propose that the enzyme discriminates against glucose by favouring interaction with the non-reducing end of cellobiose.

  • 10. Hallberg, B. M.
    et al.
    Henriksson, Gunnar
    KTH, Superseded Departments, Pulp and Paper Technology.
    Pettersson, G.
    Vasella, A.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Mechanism of the reductive half-reaction in cellobiose dehydrogenase2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 9, p. 7160-7166Article in journal (Refereed)
    Abstract [en]

    The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC 1.1.99.18) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DHcdh) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DHcdh catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DHcdh is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DHcdh with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-Angstrom resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 Angstrom, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8-0.9 Angstrom. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5.

  • 11.
    Hallberg, Martin
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Leitner, C.
    Haltrich, D.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 341, no 3, p. 781-796Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8 Angstrom resolution to R and R-free values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three U helices. The homotetramer buries a large internal cavity of roughly 15,000 Angstrom(3), from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.

  • 12.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Geiger, Barbara
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
    Patel, Bharat K C
    Kittl, Roman
    Haltrich, Dietmar
    Nguyen, Thu-Ha
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
    Engineering a polyextremophilic Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesisManuscript (preprint) (Other academic)
  • 13.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Geiger, Barbara
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Patel, Bharat K. C.
    Kittl, Roman
    Haltrich, Dietmar
    Nguyen, Thu-Ha
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 8, p. 3533-3543Article in journal (Refereed)
    Abstract [en]

    Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining beta-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a beta-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k(cat), but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k(cat)). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with similar to 10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

  • 14.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
    Kori, Lokesh D
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
    Patel, Bharat K C
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
    High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with alpha-L-arabinofuranosidase activity2015In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 71, no Pt 3, p. 338-45Article in journal (Refereed)
    Abstract [en]

    A gene from the heterotrophic, halothermophilic marine bacterium Halothermothrix orenii has been cloned and overexpressed in Escherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by H. orenii. The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10Å resolution. As for other GH43 members, the enzyme folds as a five-bladed β-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, the H. orenii glycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4M NaCl and Mn2+ at pH 6.5. The gene encoding the H. orenii GH43 enzyme has previously been annotated as a putative α-l-arabinofuranosidase. Activity was detected with p-nitrophenyl-α-l-arabinofuranoside as a substrate, and therefore the name HoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4M NaCl and Mn2+ at pH 6.5, giving a specific activity of 20-36μmolmin-1mg-1. The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution of HoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.

  • 15.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO).
    Nguyen, Thu-Ha
    Intanon, Montira
    Kori, Lokesh D.
    Patel, Bharat K. C.
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO).
    Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis2015In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 99, no 4, p. 1731-1744Article in journal (Refereed)
    Abstract [en]

    Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific beta-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-D-Galp-(1 -> 3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

  • 16.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tan, T. -C
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Pisanelli, I.
    Fusco, L.
    Haltrich, D.
    Peterbauer, C. K.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly2013In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 3, p. 496-504Article in journal (Refereed)
    Abstract [en]

    The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-. d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-. β-. d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17°C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.

  • 17. Henriksson, G
    et al.
    Salumets, A
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Pettersson, G
    Studies of cellulose binding by cellobiose dehydrogenase and a comparison with cellobiohydrolase 11997In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 324, p. 833-838Article in journal (Refereed)
  • 18. Henriksson, H
    et al.
    Stahlberg, J
    Koivula, A
    Pettersson, G
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Valtcheva, L
    Isaksson, R
    The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition1997In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 57, no 1-3, p. 115-125Article in journal (Refereed)
  • 19.
    Hällberg, B. Martin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Leitner, Christian
    KTH, Superseded Departments, Biotechnology.
    Haltrich, Dietmar
    KTH, Superseded Departments, Biotechnology.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Crystallization and preliminary X-ray diffraction analysis of pyranose 2-oxidase from the white-rot fungus Trametes multicolor2004In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 60, p. 197-199Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetrameric flavoenzyme that catalyzes the oxidation of D-glucose to 2-keto-D-glucose. P2Ox participates in lignin degradation by white-rot fungi and a tentative role of the enzyme is the production of H2O2 for lignin peroxidases. Crystals of Trametes multicolor P2Ox were grown from monomethylether PEG 2000, sodium acetate, MgCl2 and Ta6Br12. They belong to space group P2(1), with unit-cell parameters a = 99.9, b = 101.7, c = 135.6 Angstrom, beta = 90.85degrees. X-ray diffraction data to 2.0 Angstrom resolution were collected using synchrotron radiation. Self-rotation function calculations suggest that the asymmetric unit contains one homotetramer with 222 point-group symmetry.

  • 20. Kittl, Roman
    et al.
    Sygmund, Christoph
    Halada, Petr
    Volc, Jindrich
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Peterbauer, Clemens K.
    Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR2008In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 53, no 2, p. 117-127Article in journal (Refereed)
    Abstract [en]

    Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation.

  • 21. Kleywegt, G J
    et al.
    Zou, J Y
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Davies, G J
    Sinning, I
    Stahlberg, J
    Reinikainen, T
    Srisodsuk, M
    Teeri, T T
    Jones, T A
    The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 angstrom resolution, and a comparison with related enzymes1997In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 272, no 3, p. 383-397Article in journal (Refereed)
  • 22. Kujawa, M
    et al.
    Leitner, C
    Halada, P
    Volc, J
    Hallberg, B M
    Divne, Christina
    Peterbauer, C K
    Haltrich, D
    Pyranose oxidase from Trametes multicolour: application in biocatalysis2005In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 118, p. 89-89Article in journal (Refereed)
  • 23. Kujawa, Magdalena
    et al.
    Ebner, Heidemarie
    Leitner, Christian
    Hallberg, B. Martin
    Prongjit, Methinee
    Sucharitakul, Jeerus
    Ludwig, Roland
    Rudsander, Ulla
    Peterbauer, Clemens
    Chaiyen, Pimchai
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 46, p. 35104-35115Article in journal (Refereed)
    Abstract [en]

    Pyranose2-oxidase(P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regios-electivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k(cat), and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp(452) in the substrate loop and, possibly, a nearby arginine residue (Arg(472)). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp(452) and Arg(472) as well as the double alanine 452/472 mutant.

  • 24. Kujawa, Magdalena
    et al.
    Volc, Jindrich
    Halada, Petr
    Sedmera, Petr
    Divne, Christina
    Univ Nat & Appl Life Sci, Austria.
    Sygmund, Christian
    Peterbauer, Leitner Clemens
    Haltrich, Dietmar
    Properties of pyranose dehydrogenase purified from Agaricus xanthoderma2007Conference paper (Refereed)
  • 25. Kujawa, Magdalena
    et al.
    Volc, Jindrich
    Halada, Petr
    Sedmera, Petr
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sygmund, Christoph
    Leitner, Christian
    Peterbauer, Clemens
    Haltrich, Dietmar
    Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 3, p. 879-894Article in journal (Refereed)
    Abstract [en]

    We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K-m). The enzyme may play a role in lignocellulose degradation.

  • 26. Mackenzie, L F
    et al.
    Sulzenbacher, G
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Jones, T A
    Woldike, H F
    Schulein, M
    Withers, S G
    Davies, G J
    Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 angstrom resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate1998In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 335, p. 409-416Article in journal (Refereed)
  • 27. Mason, M. G.
    et al.
    Nicholls, P.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Hallberg, B. M.
    Henriksson, Gunnar
    KTH, Superseded Departments, Pulp and Paper Technology.
    Wilson, M. T.
    The heme domain of cellobiose oxidoreductase: a one-electron reducing system2003In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1604, no 1, p. 47-54Article in journal (Refereed)
    Abstract [en]

    Phanerochaete chrysosporium cellobiose oxidoreductase (CBOR) comprises two redox domains, one containing flavin adenine dinucleotide (FAD) and the other protoheme. It reduces both two-electron acceptors, including molecular oxygen, and one-electron acceptors, including transition metal complexes and cytochrome c. If the latter reacts with the flavin, the reduced heme b acts merely as a redox buffer, but if with the b heme, enzyme action involves a true electron transfer chain. Intact CBOR fully reduced with cellobiose, CBOR partially reduced by ascorbate, and isolated ascorbate-reduced heme domain, all transfer electrons at similar rates to cytochrome c. Reduction of cationic one-electron acceptors via the heme group supports an electron transfer chain model. Analogous reactions with natural one-electron acceptors can promote Fenton chemistry, which may explain evolutionary retention of the heme domain and the enzyme's unique character among secreted sugar dehydrogenases.

  • 28.
    Master, Emma
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Rudsander, Ulla
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Zhou, Welin
    Henriksson, Hongbin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Divne, Christina
    KTH, Superseded Departments (pre-2005), Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Denman, Stuart
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Wilson, David
    Teeri, Tuula
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 31, p. 10080-10089Article in journal (Refereed)
    Abstract [en]

    PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30degreesC and pH 6.0, the k(cat) for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (k(cat)/K-m) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.

  • 29. Nguyen, Thu-Ha
    et al.
    Splechtna, Barbara
    Krasteva, Stanimira
    Kneifel, Wolfgang
    Kulbe, Klaus D.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Haltrich, Dietmar
    Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R222007In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 269, no 1, p. 136-144Article in journal (Refereed)
    Abstract [en]

    beta-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The K-m and V-max values for lactose and oNPG were 4.04 +/- 0.26 mM, 28.8 +/- 0.2 mu mol D-glucose released per min per mg protein, and 0.73 +/- 0.07 mM, 361 +/- 12 mu mol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with K-i,K-s=31.7 +/- 3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. beta-Galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.

  • 30. Pitsawong, Warintra
    et al.
    Sucharitakul, Jeerus
    Prongjit, Methinee
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Chaiyen, Pimchai
    A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 13, p. 9697-9705Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase (P2O) catalyzes the oxidation by O-2 of D-glucose and several aldopyranoses to yield the 2-ketoaldoses and H2O2. Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr(169) forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O center dot sugar complex. Transient kinetics of wild-type (WT) and Thr(169)-> S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K-d (45-47 mM), and the hydride transfer rate constants (k(red)) are similar (15.3-9.7 s(-1) at 4 degrees C). k(red) of T169G with D-glucose (0.7 s(-1), 4 degrees C) is significantly less than that of WT but not as severely affected as in T169A (k(red) of 0.03 s(-1) at 25 degrees C). Transient kinetics of WT and mutants using D-galactose show that P2O binds D-galactose with a one-step binding process, different from binding of D- glucose. In T169S, T169N, and T169G, the overall turnover with D- Gal is faster than that of WT due to an increase of kred. In the crystal structure of T169S, Ser(169) O gamma assumes a position identical to that of O gamma 1 in Thr(169); in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr(169) side chain are required for intermediate stabilization.

  • 31.
    Quehenberger, Julian
    et al.
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Reichenbach, Tom
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Baumann, Niklas
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Rettenbacher, Lukas
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Spadiut, Oliver
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum.2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 1, article id E185Article in journal (Refereed)
    Abstract [en]

    While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

  • 32. RAICES, M
    et al.
    PAIFER, E
    CREMATA, J
    MONTESINO, R
    STAHLBERG, J
    DIVNE, Christina
    Uppsala University, Sweden.
    SZABO, IJ
    HENRIKSSON, G
    JOHANSSON, G
    PETTERSSON, G
    Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium1995In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 369, no 2-3, p. 233-238Article in journal (Refereed)
    Abstract [en]

    The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5′ end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide-binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.

  • 33.
    Rajangam, Alex S.
    et al.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Kumar, Manoj
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Guerriero, Gea
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Pansri, Podjamas
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Brown, Christian J. L.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Blomqvist, Kristina
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Mellerowicz, Ewa
    Sundberg, Bjorn
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    MAP20, a Microtubule-Associated Protein in the Secondary Cell Walls of Hybrid Aspen, Is a Target of the Cellulose Synthesis Inhibitor 2,6-Dichlorobenzonitrile2008In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 148, no 3, p. 1283-1294Article in journal (Refereed)
    Abstract [en]

    We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

  • 34.
    Reichenbach, Tom
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Kalyani, Dayanand
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gandini, Rosaria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svartström, Olov
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Aspeborg, Henrik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Structural and biochemical characterization of the Cutibacterium acnes exo-β-1,4-mannosidase that targets the N-glycan core of host glycoproteins.2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 9, article id e0204703Article in journal (Refereed)
    Abstract [en]

    Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host's immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-β-1,4-mannosidase with activity on the β-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway.

  • 35. Rotsaert, F. A. J.
    et al.
    Hallberg, B. M.
    de Vries, S.
    Moenne-Loccoz, P.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Renganathan, V.
    Gold, M. H.
    Biophysical and structural analysis of a novel heme b iron ligation in the flavocytochrome cellobiose dehydrogenase2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 35, p. 33224-33231Article in journal (Refereed)
    Abstract [en]

    The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met(65) was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Angstrom resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N-delta1 and N-epsilon2 nitrogen atoms, rather than the typical N-epsilon2/N-epsilon2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N-delta1/N-epsilon2-coordinated protoporphyrin IX iron.

  • 36. Salaheddin, Clara
    et al.
    Spadiut, Oliver
    Ludwig, Roland
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Peterbauer, Clemens
    Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design.2009In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 4, no 4, p. 535-543Article in journal (Refereed)
    Abstract [en]

    D-Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be produced by an enzymatic oxidation of D-galactose specifically at C2 followed by chemical hydrogenation. Pyranose 2-oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. Since D-galactose is not the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the active site of the enzyme, resulting in a library of mutants, which were screened for improved activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold increase of k(cat) with both D-glucose and D-galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, k(cat)/K(M) is lower for both sugar substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e., T169S and T169N, showed an improvement of the catalytic characteristics of P2Ox with D-galactose. Batch conversion experiments of D-galactose to 2-keto-D-galactose were performed with wild-type TmP2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis-Menten kinetics.

  • 37. Spadiut, Oliver
    et al.
    Leitner, Christian
    Salaheddin, Clara
    Varga, Balazs
    Vertessy, Beata G.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 3, p. 776-792Article in journal (Refereed)
    Abstract [en]

    The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 degrees C, respectively, relative to the wild-type enzyme. Although both wild-type and single mutants showed first-order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association.

  • 38. Spadiut, Oliver
    et al.
    Leitner, Christian
    Tan, Tien-Chye
    Ludwig, Roland
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Mutations of Thr169 affect substrate specificity of pyranose 2-oxidase from Trametes multicolor2008In: Biocatalysis and Biotransformation, ISSN 1024-2422, E-ISSN 1029-2446, Vol. 26, no 1-2, p. 120-127Article in journal (Refereed)
    Abstract [en]

    Site-directed mutagenesis was used to enhance the catalytic activity of pyranose 2-oxidase (P2Ox) from Trametes multicolor with different substrates. To this end, threonine at position 169 was replaced by glycine, alanine and serine, respectively. Using oxygen as electron acceptor the mutant T169G was equally active with d-glucose and d-galactose, whereas wild-type recombinant P2Ox only showed 5.2% relative activity with the latter substrate. When d-galactose was used as electron donor in saturating concentrations, T169G showed a 4.5-fold increase in its catalytic efficiency k(cat)/K-M for the alternative electron acceptor 1,4-benzoquinone and a nine-fold increased k(cat)/K-M value with the ferricenium ion compared with wt recP2Ox. Variant T169S showed an increase in its catalytic efficiency both with 1,4-benzoquinone (3.7 times) as well as with the ferricenium ion (1.4 times) when D-glucose was the substrate.

  • 39. Spadiut, Oliver
    et al.
    Radakovits, Katrin
    Pisanelli, Ines
    Salaheddin, Clara
    Yamabhai, Montarop
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications.2009In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 4, no 4, p. 525-534Article in journal (Refereed)
    Abstract [en]

    In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 172], the variant oxidizes both sugars equally well [(k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K(M) values and approximately ten-fold higher k(cat) values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60 degrees C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D-glucose/D-galactose mixtures at both 30 and 50 degrees C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 A resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.

  • 40.
    Spadiut, Oliver
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Biochemistry.
    Pisanelli, Ines
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry.
    Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 13, p. 2892-2909Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment (454FSY456) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp452, Phe454 and Tyr456) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.

  • 41. Stahlberg, J
    et al.
    Divne, Christina
    University of Uppsala, Sweden.
    Koivula, A
    Piens, K
    Claeyssens, M
    Teeri, T T
    Jones, T A
    Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei1996In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 264, no 2, p. 337-349Article in journal (Refereed)
  • 42. Stahlberg, J.
    et al.
    Henriksson, H.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Isaksson, R.
    Pettersson, G.
    Johansson, G.
    Jones, T. A.
    Structural basis for enantiomer binding and separation of a common beta-blocker: Crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 angstrom resolution2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 305, no 1, p. 79-93Article in journal (Refereed)
    Abstract [en]

    Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta -blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers. In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta -blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1.9 Angstrom resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/ + 1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O-epsilon1 and to Gln175 N-epsilon2, whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q. Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 muM and 44 muM, respectively, corresponding to binding free energies of 20 kJ/ mol and 24 kJ/mol, respectively. The K-i value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed. By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non-selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.

  • 43.
    Tan, Tien Chye
    et al.
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gandini, Rosaria
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, article id e86736Article in journal (Refereed)
    Abstract [en]

    Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/ V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the a-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the beta-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the beta-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose alpha-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.

  • 44.
    Tan, Tien Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wongnate, T.
    Sucharitakul, J.
    Krondorfer, I.
    Sygmund, C.
    Haltrich, D.
    Chaiyen, P.
    Peterbauer, C. K.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1Article in journal (Refereed)
    Abstract [en]

    Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.

  • 45.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Regioselective Control of beta-D-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, no 4, p. 588-600Article in journal (Refereed)
    Abstract [en]

    Trametes multicolor pyranose 2-oxidase (P2O) is a flavoprotein oxidase that oxidizes D-glucose at C2 to 2-keto-D-glucose by a highly regioselective mechanism. In this work, fluorinated sugar substrates were used as mechanistic probes to investigate the basis of regioselectivity in P2O. Although frequently used to study the mechanisms of glycoside hydrolases, our work provides the first example of applying these probes to sugar oxidoreductases. Our previous structure of the P2O mutant H167A in complex with the slow substrate 2-deoxy-2-fluoro-D-glucose showed a substrate-binding mode compatible with oxidation at C3. To accommodate the sugar, a gating segment, (FSY456)-F-454, in the substrate recognition loop partly unfolded to create a spacious and more polar active site that is distinct from the closed state of P2O. The crystal structure presented here shows that the preferred C2 oxidation where an ordered complex of P2O H167A with 3-deoxy-3-fluoro-D-glucose at 1.35 angstrom resolution was successfully trapped. In this semi-open C2-oxidation complex, the substrate recognition loop tightens to form an optimized substrate complex stabilized by interactions between Asp452 and glucose O4, as well as Tyr456 and the glucose O6 group, interactions that are not possible when glucose is positioned for oxidation at C3. The different conformations of the (FSY456)-F-454 gating segment in the semiopen and closed states induce backbone and side-chain movements of Thr169 and Asp452 that add further differential stabilization to the individual states. We expect the semi-open state (C2-oxidation state) and closed state to be good approximations of the active-site structure during the reductive half-reaction (sugar oxidation) and oxidative half-reaction (O-2 reduction).

  • 46.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Kracher, Daniel
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sygmund, Christoph
    Kittl, Roman
    Haltrich, Dietmar
    Hallberg, B Martin
    Ludwig, Roland
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, p. 7542-7542Article in journal (Refereed)
    Abstract [en]

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

  • 47. Tan, Tien-Chye
    et al.
    Mijts, Benjamin N.
    Swaminathan, Kunchithapadam
    Patel, Bharat K. C.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: Details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 378, no 4, p. 852-870Article in journal (Refereed)
    Abstract [en]

    The gene for a membrane-bound, halophilic, and thermostable alpha-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove-the N-C groove some 30 angstrom away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 angstrom resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with alpha-cyclodextrin and maltoheptaose at 2.2 angstrom resolution, a maltotetraose molecule is bound in subsites -1 to +3, spanning the cleavage point at -1 / + 1, with the -1 glucosyl residue present as a S-2(o) skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across -1/+ 1 and ring distortion. We observe three methionine side chains that serve as '' binding platforms '' for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.

  • 48.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Pitsawong, Warintra
    Wongnate, Thanyaporn
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Haltrich, Dietmar
    Chaiyen, Pimchai
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 402, no 3, p. 578-594Article in journal (Refereed)
    Abstract [en]

    Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond-the flavinylation reaction-has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand. (c) 2010 Elsevier Ltd. All rights reserved.

  • 49. Teeri, T T
    et al.
    Koivula, A
    Linder, M
    Wohlfahrt, G
    Divne, Christina
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Jones, T. A.
    Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?1998Conference paper (Refereed)
  • 50. TEERI, TT
    et al.
    KOIVULA, A
    REINIKAINEN, T
    RUOHONEN, L
    SRISODSUK, M
    DIVNE, Christina
    Uppsala University, Sweden.
    ROUVINEN, J
    SZARDENINGS, M
    JONES, TA
    HYDROLYSIS OF CRYSTALLINE CELLULOSE BY NATIVE AND ENGINEERED TRICHODERMA-REESEI CELLULASES1994In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 207, p. 21-AGFDArticle in journal (Refereed)
12 1 - 50 of 59
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