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  • 1.
    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)
  • 2.
    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.

  • 3.
    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)
  • 4.
    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)
  • 5.
    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.

  • 6.
    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.

  • 7.
    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.

  • 8. 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.

  • 9. 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.

  • 10. 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.

  • 11. 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.

  • 12.
    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.

  • 13.
    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.

  • 14.
    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.

  • 15.
    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).

  • 16.
    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.

  • 17.
    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.

  • 18. Ullmann, Eva
    et al.
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO). Karolinska Institutet, Stockholm, Sweden .
    Gundinger, Thomas
    Herwig, Christoph
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Karolinska Institutet, Stockholm, Sweden .
    Spadiut, Oliver
    A novel cytosolic NADH: quinone oxidoreductase from Methanothermobacter marburgensis2014In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 34, p. 893-904Article in journal (Refereed)
    Abstract [en]

    Methanothermobacter marburgensis is a strictly anaerobic, thermophilic methanogenic archaeon that uses methanogenesis to convert H-2 and CO2 to energy. M. marburgensis is one of the best-studied methanogens, and all genes required for methanogenic metabolism have been identified. Nonetheless, the present study describes a gene (Gene ID 9704440) coding for a putative NAD(P)H:quinone oxidoreductase that has not yet been identified as part of the metabolic machinery. The gene product, MmNQO, was successfully expressed, purified and characterized biochemically, as well as structurally. MmNQO was identified as a flavin-dependent NADH: quinone oxidoreductase with the capacity to oxidize NADH in the presence of a wide range of electron acceptors, whereas NADPH was oxidized with only three acceptors. The 1.50 angstrom crystal structure of MmNQO features a homodimeric enzyme where each monomer comprises 196 residues folding into flavodoxin-like alpha/beta domains with non-covalently bound FMN (flavin mononucleotide). The closest structural homologue is the modulator of drug activity B from Streptococcus mutans with 1.6 angstrom root-mean-square deviation on 161 C alpha atoms and 28% amino-acid sequence identity. The low similarity at sequence and structural level suggests that MmNQO is unique among NADH: quinone oxidoreductases characterized to date. Based on preliminary bioreactor experiments, MmNQO could provide a useful tool to prevent overflow metabolism in applications that require cells with high energy demand.

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