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Hassan, N., Nguyen, T.-H., Intanon, M., Kori, L. D., Patel, B. K. C., Haltrich, D., . . . Tan, T. C. (2015). Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis. Applied Microbiology and Biotechnology, 99(4), 1731-1744
Open this publication in new window or tab >>Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis
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2015 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 99, no 4, p. 1731-1744Article in journal (Refereed) Published
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.

Keywords
beta-glucosidase, beta-galactosidase, Halothermophile, Halothermothrix, Lactose conversion, Galacto-oligosaccharides, Biochemical characterization, Structural analysis
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-162963 (URN)10.1007/s00253-014-6015-x (DOI)000350028600017 ()25173693 (PubMedID)2-s2.0-84922434915 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150331

Available from: 2015-03-31 Created: 2015-03-26 Last updated: 2017-12-04Bibliographically approved
Wang, Y., Azhar, S., Gandini, R., Divne, C., Ezcurra, I. & Aspeborg, H. (2015). Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana. Plant Science, 241, 151-163
Open this publication in new window or tab >>Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana
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2015 (English)In: Plant Science, ISSN 0168-9452, E-ISSN 1873-2259, Vol. 241, p. 151-163Article in journal (Refereed) Published
Abstract [en]

Plant mannanases are enzymes that carry out fundamentally important functions in cell wall metabolism during plant growth and development by digesting manno-polysaccharides. In this work, the Arabidopsis mannanase 5-2 (AtMan5-2) from a previously uncharacterized subclade of glycoside hydrolase family 5 subfamily 7 (GH5_7) has been heterologously produced in Pichia pastoris. Purified recombinant AtMan5-2 is a glycosylated protein with an apparent molecular mass of 50 kDa, a pH optimum of 5.5-6.0 and a temperature optimum of 25 degrees C. The enzyme exhibits high substrate affinity and catalytic efficiency on mannan substrates with main chains containing both glucose and mannose units such as konjac glucomannan and spruce galactoglucomannan. Product analysis of manno-oligosaccharide hydrolysis shows that AtMan5-2 requires at least six substrate-binding subsites. No transglycosylation activity for the recombinant enzyme was detected in the present study. Our results demonstrate diversification of catalytic function among members in the Arabidopsis GH5_7 subfamily.

Place, publisher, year, edition, pages
Elsevier, 2015
Keywords
Glycoside hydrolase, GH5, endo-β-1, 4-Mannan hydrolase, Cell wall, Mannan polysaccharides/oligosaccharides
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-178198 (URN)10.1016/j.plantsci.2015.10.002 (DOI)000367487500015 ()2-s2.0-84945291912 (Scopus ID)
Funder
Swedish Foundation for Strategic Research VINNOVASwedish Research Council Formas
Note

QC 20160104. QC 20160201

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Hassan, N., Kori, L. D., Gandini, R., Patel, B. K., Divne, C. & Tan, T. C. (2015). High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with alpha-L-arabinofuranosidase activity. Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, 71(Pt 3), 338-45
Open this publication in new window or tab >>High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with alpha-L-arabinofuranosidase activity
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2015 (English)In: 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) Published
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.

Place, publisher, year, edition, pages
International Union of Crystallography, 2015
Keywords
glycoside hydrolase, five-bladed beta-propeller, arabinofuranosidase, Halothermothrix orenii, halothermophile
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-164102 (URN)10.1107/S2053230X15003337 (DOI)000351157900016 ()25760712 (PubMedID)2-s2.0-84924655292 (Scopus ID)
Funder
Swedish Research Council Formas, 2013-1741Swedish Research Council, 2013-5717
Note

QC 20150419

Available from: 2015-04-13 Created: 2015-04-13 Last updated: 2017-12-04Bibliographically approved
Tan, T.-C., Kracher, D., Gandini, R., Sygmund, C., Kittl, R., Haltrich, D., . . . Divne, C. (2015). Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation. Nature Communications, 6, 7542-7542
Open this publication in new window or tab >>Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, p. 7542-7542Article in journal (Refereed) Published
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.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-171174 (URN)10.1038/ncomms8542 (DOI)000358852700001 ()26151670 (PubMedID)2-s2.0-84936851753 (Scopus ID)
Funder
Swedish Research Council Formas, 2008-495Swedish Research Council Formas, 2013-1741Swedish Research Council, 2008-4056Swedish Research Council, 2011-5768Swedish Research Council, 2011-6510Carl Tryggers foundation , CTS08:78EU, FP7, Seventh Framework Programme, FP7-KBBE-2013-7-613549
Note

QC 20150720

Available from: 2015-07-20 Created: 2015-07-20 Last updated: 2017-12-04Bibliographically approved
Ullmann, E., Tan, T. C., Gundinger, T., Herwig, C., Divne, C. & Spadiut, O. (2014). A novel cytosolic NADH: quinone oxidoreductase from Methanothermobacter marburgensis. Bioscience Reports, 34, 893-904
Open this publication in new window or tab >>A novel cytosolic NADH: quinone oxidoreductase from Methanothermobacter marburgensis
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2014 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 34, p. 893-904Article in journal (Refereed) Published
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.

Keywords
crystal structure, cytoplasm, Methanothermobacter marburgensis, NADH regeneration, NADH:quinone oxidoreductase
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-160011 (URN)10.1042/BSR20140143 (DOI)000347799400021 ()2-s2.0-84920126801 (Scopus ID)
Note

QC 20150216

Available from: 2015-02-16 Created: 2015-02-12 Last updated: 2017-12-04Bibliographically approved
Tan, T. C., Spadiut, O., Gandini, R., Haltrich, D. & Divne, C. (2014). Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion. PLoS ONE, 9(1), e86736
Open this publication in new window or tab >>Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, p. e86736-Article in journal (Refereed) Published
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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-141963 (URN)10.1371/journal.pone.0086736 (DOI)000330244500278 ()2-s2.0-84907020984 (Scopus ID)
Funder
Swedish Research Council, 2008-4045 2011-5768
Note

QC 20140227

Available from: 2014-02-27 Created: 2014-02-27 Last updated: 2017-12-05Bibliographically approved
Hassan, N., Tan, T.-C. -., Spadiut, O., Pisanelli, I., Fusco, L., Haltrich, D., . . . Divne, C. (2013). Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly. FEBS Open Bio, 3, 496-504
Open this publication in new window or tab >>Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly
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2013 (English)In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 3, p. 496-504Article in journal (Refereed) Published
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.

Keywords
Crystal structure, Lignin degradation, Oligomerization, Propeptide, Pyranose 2-oxidase, Thermostability
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-139900 (URN)10.1016/j.fob.2013.10.010 (DOI)000339569800078 ()2-s2.0-84888118395 (Scopus ID)
Funder
Swedish Research Council, 2008-4045 2011-5768
Note

QC 20140115

Available from: 2014-01-15 Created: 2014-01-15 Last updated: 2017-12-06Bibliographically approved
Tan, T. C., Spadiut, O., Wongnate, T., Sucharitakul, J., Krondorfer, I., Sygmund, C., . . . Divne, C. (2013). The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate. PLoS ONE, 8(1), e53567
Open this publication in new window or tab >>The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1, p. e53567-Article in journal (Refereed) Published
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.

Keywords
Aryl-Alcohol Oxidase, Cellobiose Dehydrogenase, Flavoprotein Oxidases, Oxygen Activation, Protein-Structure, Hydride Transfer, Glucose-Oxidase, Choline Oxidase, C-3 Oxidation, Active-Site
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-118222 (URN)10.1371/journal.pone.0053567 (DOI)000313551500085 ()2-s2.0-84872224409 (Scopus ID)
Funder
FormasSwedish Research Council
Note

QC 20130213

Available from: 2013-02-13 Created: 2013-02-13 Last updated: 2017-12-06Bibliographically approved
Tan, T.-C., Haltrich, D. & Divne, C. (2011). Regioselective Control of beta-D-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy. Journal of Molecular Biology, 409(4), 588-600
Open this publication in new window or tab >>Regioselective Control of beta-D-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy
2011 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, no 4, p. 588-600Article in journal (Refereed) Published
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).

Keywords
Pyranose 2-oxidase, conformational degeneracy, regioselectivity, substrate specificity, glucose oxidation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-36234 (URN)10.1016/j.jmb.2011.04.019 (DOI)000292175100010 ()
Note
QC 20110711Available from: 2011-07-11 Created: 2011-07-11 Last updated: 2017-12-11Bibliographically approved
Pitsawong, W., Sucharitakul, J., Prongjit, M., Tan, T.-C., Spadiut, O., Haltrich, D., . . . Chaiyen, P. (2010). A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase. Journal of Biological Chemistry, 285(13), 9697-9705
Open this publication in new window or tab >>A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase
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2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 13, p. 9697-9705Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-28373 (URN)10.1074/jbc.M109.073247 (DOI)000276165900042 ()2-s2.0-77951245033 (Scopus ID)
Funder
FormasSwedish Research Council
Note
QC 20110119Available from: 2011-01-19 Created: 2011-01-14 Last updated: 2017-12-11Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-5805-2693

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