Thermobifida fusca

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David Wilson - One of the best experts on this subject based on the ideXlab platform.

  • Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues.
    Biotechnology for biofuels, 2017
    Co-Authors: Nathan Kruer-zerhusen, Markus Alahuhta, Vladimir V. Lunin, Michael E. Himmel, Yannick J. Bomble, David Wilson
    Abstract:

    Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The point mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.

  • Processivity, synergism, and substrate specificity of Thermobifida fusca Cel6B.
    Applied and environmental microbiology, 2009
    Co-Authors: Thu V. Vuong, David Wilson
    Abstract:

    A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.

  • The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B
    The FEBS journal, 2009
    Co-Authors: Thu V. Vuong, David Wilson
    Abstract:

    Thermobifida fusca exocellulase Cel6B acts by an inverting hydrolysis mechanism; however, the catalytic acid and base residues for this enzyme have not been confirmed. Site-directed mutagenesis and kinetic studies were used to show that Asp274 is the catalytic acid, which is consistent with what is found for other members of family-6 glycoside hydrolases; however, a single catalytic base was not identified. Mutation of all putative catalytic base residues, within 6 A of the −1/+1 glucose subsites, including the highly conserved Asp226, Asp497 and Glu495, as well as Ser232 and Tyr220, did not reveal a catalytic base, although these residues are all important for activity. We propose a novel hydrolysis mechanism for T. fusca Cel6B involving a proton-transferring network to carry out the catalytic base function.

  • Chitin binding by Thermobifida fusca cellulase catalytic domains.
    Biotechnology and bioengineering, 2008
    Co-Authors: David Wilson
    Abstract:

    Cellulose is a linear homopolymer of b1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to a-chitin was investigated. The CDs of endocel- lulases, Cel6A and Cel5A did not bind to a-chitin; one exocellulase, Cel48A CD bound a-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD boundextremely tightly to a-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to a-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked success- fully to the catalytic cleft ofCel9A. In general, the positioningof the sugar residues in the model structures matched the cello- oligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose. Biotechnol. Bioeng. 2008;XXX: XXX-XXX. 2008 Wiley Periodicals, Inc.

  • Comparative NMR analysis of cello-oligosaccharide hydrolysis by family GH9 endoglucanases from Thermobifida fusca and Populus tremula x tremuloides Mich
    Biochemistry, 2007
    Co-Authors: Ulla Rudsander, David Wilson, Corine Sandström, Kathleen Piens, Emma R. Master, Harry Brumer, Lennart Kenne, Tuula T. Teeri
    Abstract:

    Comparative NMR analysis of cello-oligosaccharide hydrolysis by family GH9 endoglucanases from Thermobifida fusca and Populus tremula x tremuloides Mich

Marco W. Fraaije - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of a chitinase from the cellulolytic actinomycete Thermobifida fusca.
    Biochimica et biophysica acta, 2016
    Co-Authors: Yasser Gaber, Sophanit Mekasha, Gustav Vaaje-kolstad, Vincent G. H. Eijsink, Marco W. Fraaije
    Abstract:

    Abstract Thermobifida fusca is a well-known cellulose-degrading actinomycete, which produces various glycoside hydrolases for this purpose. However, despite the presence of putative chitinase genes in its genome, T. fusca has not been reported to grow on chitin as sole carbon source. In this study, a gene encoding a putative membrane-anchored GH18 chitinase (Tfu0868) from T. fusca has been cloned and overexpressed in Escherichia coli . The protein was produced as SUMO fusion protein and, upon removal of the SUMO domain, soluble pure Tf Chi18A was obtained with yields typically amounting to 150 mg per litre of culture. The enzyme was found to be relatively thermostable (apparent T m  = 57.5 °C) but not particularly thermoactive, the optimum temperature being 40–45 °C. Tf Chi18A bound to α- and β-chitin and degraded both these substrates. Interestingly, activity towards colloidal chitin was minimal and in this case, substrate inhibition was observed. Tf Chi18A also cleaved soluble chito-oligosaccharides and showed a clear preference for substrates having five sugars or more. While these results show that Tf Chi18A is a catalytically competent GH18 chitinase, the observed catalytic rates were low compared to those of well-studied GH18 chitinases. This suggests that Tf Chi18A is not a true chitinase and not likely to endow T. fusca with the ability to grow on chitin.

  • Exploring the biocatalytic potential of a DyP-type peroxidase by profiling the substrate acceptance of Thermobifida fusca DyP peroxidase
    Tetrahedron, 2016
    Co-Authors: Nikola Lončar, Dana I. Colpa, Marco W. Fraaije
    Abstract:

    Dye-decolorizing peroxidases (DyPs) represent a new class of oxidative enzymes for which the natural substrates are largely unknown. To explore the biocatalytic potential of a DyP, we have studied the substrate acceptance profile of a robust DyP peroxidase, a DyP from Thermobifida fusca (TfuDyP). While previous work established that this bacterial peroxidase is able to act on a few reactive dyes and aromatic sulfides, this work significantly expands its substrate scope towards lignin related compounds, flavors, and various dyes. (C) 2016 Elsevier Ltd. All rights reserved.

  • Characterization of a chitinase from the cellulolytic actinomycete Thermobifida fusca Proteins and proteomics
    Biochimica et Biophysica Acta, 2016
    Co-Authors: Yasser Gaber, Sophanit Mekasha, Gustav Vaaje-kolstad, Vincent G. H. Eijsink, Marco W. Fraaije
    Abstract:

    Thermobifida fusca is a well-known cellulose-degrading actinomycete, which produces various glycoside hydrolases for this purpose. However, despite the presence of putative chitinase genes in its genome, T. fusca has not been reported to grow on chitin as sole carbon source. In this study, a gene encoding a putative membrane-anchored GH18 chitinase (Tfu0868) from T. fusca has been cloned and overexpressed in Escherichia coli. The protein was produced as SUMO fusion protein and, upon removal of the SUMO domain, soluble pure TfChi18A was obtained with yields typically amounting to 150mg per litre of culture. The enzyme was found to be relatively thermostable (apparent Tm=57.5°C) but not particularly thermoactive, the optimum temperature being 40–45°C. TfChi18A bound to α- and β-chitin and degraded both these substrates. Interestingly, activity towards colloidal chitin was minimal and in this case, substrate inhibition was observed. TfChi18A also cleaved soluble chito-oligosaccharides and showed a clear preference for substrates having five sugars or more. While these results show that TfChi18A is a catalytically competent GH18 chitinase, the observed catalytic rates were low compared to those of well-studied GH18 chitinases. This suggests that TfChi18A is not a true chitinase and not likely to endow T. fusca with the ability to grow on chitin.

  • not so monofunctional a case of thermostable Thermobifida fusca catalase with peroxidase activity
    Applied Microbiology and Biotechnology, 2015
    Co-Authors: Nikola Lončar, Marco W. Fraaije
    Abstract:

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k obs of nearly 1000 s−1 for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

  • from Thermobifida fusca
    2015
    Co-Authors: Phenylacetone Monooxygenase, Daniel Torres E. Pazmiño, Bert-jan Baas, Dick B. Janssen, Marco W. Fraaije
    Abstract:

    The Kinetic Mechanism of PAMO from Thermobifida fusca Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle (kcat = 3.1 s-1), rapid binding of NADPH (Kd = 0.7 µM) is followed by a transfer of the 4(R)-hydride from NADPH to the FAD cofactor (kred = 12 s-1). The reduced PAMO is rapidly oxygenated by molecular oxygen (kox = 870 mM-1.s-1), yielding a C4a-peroxy-flavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate (k1 = 73 s-1). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s-1) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP+ release is relatively fast and represents the final step of the catalytic cycle. Thi

David B Wilson - One of the best experts on this subject based on the ideXlab platform.

  • Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues
    BMC, 2017
    Co-Authors: Nathan Kruer-zerhusen, Markus Alahuhta, Vladimir V. Lunin, Michael E. Himmel, Yannick J. Bomble, David B Wilson
    Abstract:

    Abstract Background Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The point mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. Results In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. Conclusions The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized

  • IDENTIFICATION AND CHARACTERIZATION OF Thermobifida fusca GENES INVOLVED IN PLANT CELL WALL DEGRADATION.
    2006
    Co-Authors: David B Wilson
    Abstract:

    Micro-array experiments identified a number of Thermobifida fusca genes which were upregulated by growth on cellulose or plant biomass. Five of these genes were cloned, overexpressed in E. coli and the expressed proteins were purified and characterized. These were a xyloglucanase,a 1-3,beta glucanase, a family 18 hydrolase and twocellulose binding proteins that contained no catalytic domains. The catalyic domain of the family 74 endoxyloglucanase with a C-terminal, cellulose binding module was crystalized and its 3-dimensional structure was determined by X-ray crystallography.

  • Crystal structure of Thermobifida fusca endoglucanase Cel6A in complex with : the role of tyrosine Y73 in substrate ring distortion.
    Biochemistry, 2005
    Co-Authors: Anna M. Larsson, Diana C Irwin, David B Wilson, Terese Bergfors, Elisa Dultz, Annette K. Roos, Hugues Driguez, T. Alwyn Jones
    Abstract:

    Crystal structure of Thermobifida fusca endoglucanase Cel6A in complex with : the role of tyrosine Y73 in substrate ring distortion.

  • Purification and characterization of Thermobifida fusca xylanase 10B.
    Canadian journal of microbiology, 2004
    Co-Authors: Jeong H Kim, Diana C Irwin, David B Wilson
    Abstract:

    Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 °C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl α-D-arabinofuranoside, p-nitrophenyl-β-D-xylobioside, p-nitrophenyl-β-D- xyloside, and p-nitrophenyl-β-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.

  • Kinetic Studies of Thermobifida fusca Cel9A Active Site Mutant Enzymes
    Biochemistry, 2004
    Co-Authors: Weilin Zhou, Diana C Irwin, José M. Escovar-kousen, David B Wilson
    Abstract:

    Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding domain (Cel9A-68) followed by a fibronectin III-like domain and a family 2 cellulose binding domain. To study its catalytic mechanism, 12 mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned, and expressed in Escherichia coli. The purified mutant enzymes were assayed for their activities on (carboxymethyl)cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, and 2,4-dinitrophenyl β-d-cellobioside. They were also tested for ligand binding, enzyme processivity, and thermostability. The results clearly show that E424 functions as the catalytic acid, D55 and D58 are both required for catalytic base activity, and Y206 plays an important role in binding, catalysis, and processivity, while Y318 plays an important role in binding of crystalline cellulose substrates and is required for ...

Jian Chen - One of the best experts on this subject based on the ideXlab platform.

  • Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca.
    Bioprocess and biosystems engineering, 2013
    Co-Authors: Hui Deng, Sheng Chen, Jian Chen
    Abstract:

    Glucose isomerase (GIase) catalyzes the isomerization of d-glucose to d-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5–10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K m and K cat values of the enzyme are 197 mM and 1,688 min−1, respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.

  • Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3)
    Microbial cell factories, 2012
    Co-Authors: Sheng Zhan Chen, Ronald W. Woodard, Jian Chen
    Abstract:

    Background Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.

  • Effects of Thermobifida fusca cutinase-carbohydrate-binding module fusion proteins on cotton bioscouring
    Biotechnology and Bioprocess Engineering, 2011
    Co-Authors: Yao Zhang, Jian Chen, Sheng Chen, Qiang Wang
    Abstract:

    Previously, we presented a novel approach for increasing Thermobifida fusca cutinase adsorption on cotton fibers by fusing cutinase with a carbohydrate-binding module (CBM). A preliminary study showed that two fusion proteins, namely cutinase-CBMCel6A and cutinase-CBMCenA, with similar stabilities and catalytic properties, had potential applications in bioscouring. In the present study, an indepth analysis of both cutinase-CBMs in bioscouring was explored. Effects of cutinase-CBMs on cotton bioscouring were investigated by characterizing the chemical and physical surface changes in enzyme-treated cotton fabrics. Gas chromatography/mass spectrometry was used to analyze the degradation of the cotton fabric cuticle; Fourier transform infrared microspectroscopy was used to study changes in the chemical composition of the cotton fabric epidermal layer; and scanning electron microscopy was used to monitor minor changes in the morphology of the fiber surface. Our results indicated that cutinase-CBMs in combination with pectinase had a greater effect on cotton fabric than did cutinase. Following scouring with cutinase-CBMs and pectinase, the performance of cotton fabric in terms of its wettability and dyeability was similar to that following alkali scouring. Our study provides a foundation for the further application of cutinase-CBM to bioscouring.

  • Engineered Thermobifida fusca cutinase with increased activity on polyester substrates
    Biotechnology journal, 2011
    Co-Authors: Carla Silva, Jian Chen, Sheng Chen, Nádia Silva, Teresa Matamá, Rita Alexandra Manso Araújo, Madalena Martins, Margarida Casal
    Abstract:

    A bacterial cutinase from Thermobifida fusca, named Tfu_0883, was genetically modified by site-directed mutagenesis to enhance its activity on poly(ethylene terephthalate) (PET). The new mutations tailored the catalytic site for PET, increasing the affinity of cutinase to this hydrophobic substrate and the ability to hydrolyze it. The mutation I218A was designed to create space and the double mutation Q132A/T101A was designed both to create space and to increase hydrophobicity. The activity of the double mutant on the soluble substrate p-nitrophenyl butyrate increased two-fold compared to wild-type cutinase, while on PET both single and double mutants exhibited considerably higher hydrolysis efficiency. The replacement of specific amino acids at the active site was an effective approach for the improvement of the Tfu_0883 cutinase capacity to hydrolyze polyester surfaces. Thus, this study provides valuable insight on how the function and stability of enzymes can be improved by molecular engineering for their application in synthetic fiber biotransformation.

  • Biochemical characterization of the cutinases from Thermobifida fusca
    Journal of Molecular Catalysis B-enzymatic, 2010
    Co-Authors: Sheng Chen, Susan Billig, Wolfgang Zimmermann, Jian Chen
    Abstract:

    Abstract Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T. fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.

Alberto Boffi - One of the best experts on this subject based on the ideXlab platform.

  • Fluoride as a probe for H-bonding interactions in the active site of heme proteins: the case of Thermobifida fusca hemoglobin.
    Journal of the American Chemical Society, 2011
    Co-Authors: Francesco P. Nicoletti, Enrica Droghetti, Alessandra Bonamore, Leonardo Boechi, Darío A. Estrin, Alberto Boffi, Alessandro Feis, Natascia Sciamanna, Giulietta Smulevich
    Abstract:

    The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have...

  • Heme Pocket Structural Properties of a Bacterial Truncated Hemoglobin from Thermobifida fusca
    Biochemistry, 2010
    Co-Authors: Enrica Droghetti, Francesco P. Nicoletti, Alessandra Bonamore, Leonardo Boechi, Pau Arroyo Mañez, Darío A. Estrin, Alberto Boffi, Giulietta Smulevich, Alessandro Feis
    Abstract:

    An acidic surface variant (ASV) of the “truncated” hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level...

  • Unusual proximal heme pocket geometry in the deoxygenated Thermobifida fusca: A combined spectroscopic investigation.
    Biophysical chemistry, 2009
    Co-Authors: Alessandro Arcovito, Alessandra Bonamore, Alberto Boffi, Jean Louis Hazemann, Paola D'angelo
    Abstract:

    The spectroscopic properties of the deoxygenated truncated hemoglobin from the actinobacterium Thermobifida fusca have been investigated by means of extended X-ray absorption fine structure (EXAFS), X-ray absorption near edge structure (XANES), and near infrared spectroscopies both at room and cryogenic temperatures. At room temperature the near infrared charge transfer band III occurs at 772nm, a value that is unusually high for a canonical deoxygenated hemoglobin species, and can only be found as a transient species after photolysis in vertebrate hemoglobins and myoglobins or under strongly dehydrating conditions. EXAFS and XANES quantitative analyses, carried out in parallel with deoxygenated horse myoglobin, revealed an unusually short iron-histidine distance 1.90+/-0.03A, significantly shorter than the deoxygenated horse myoglobin distance of 2.11+/-0.02A. These findings provide novel structural basis for discussing the fine structural geometry of the proximal site, and eventually mapping the coordinates of the metal with respect to the pyrrole nitrogens and the proximal histidine nitrogen.

  • Peroxidase-like activity of Thermobifida fusca hemoglobin: The oxidation of dibenzylbutanolide
    Journal of Molecular Catalysis B-enzymatic, 2009
    Co-Authors: Roberta Torge, Alessandra Bonamore, Alessandra Comandini, Bruno Catacchio, Bruno Botta, Alberto Boffi
    Abstract:

    The thermostable truncated hemoglobin from the actinomyces Thermobifida fusca (Tf-trHb) displays a robust peroxidase activity, with optimum at acidic pH values, in experiments with the redox mediator ABTS. However, typical peroxidase substrates, such as phenolic or aromatic amine compounds, appear to be poor substrates for Tf-trHb. In turn, the protein is able to catalyze a unique dehydrogenation reaction of dibenzylbutanolides, suggested intermediates in the biosynthesis of podophyllotoxin, in the presence of hydrogen peroxide. Dibenzylbutanolides with a free 4″-hydroxyl group were thus converted into the corresponding 2,7″-dehydroderivatives thus setting up the basis for an efficient biotransformation of this important precursor. In particular, Tf-trHb mediated oxidation of trans-2-(4″-hydroxy-3″,5″-dimethoxybenzyl)-3-(3′,4′-methylenedioxy-7′β-hydroxybenzyl)butanolide 1 into the corresponding benzylidene-benzoyl-γ-butyrolactone 2 was obtained at high yield and with excellent selectivity.

  • Peroxidase-like activity of Thermobifida fusca hemoglobin: The oxidation of dibenzylbutanolide
    Journal of Molecular Catalysis B: Enzymatic, 2009
    Co-Authors: Roberta Torge, Alessandra Bonamore, Alessandra Comandini, Bruno Catacchio, Bruno Botta, Alberto Boffi
    Abstract:

    The thermostable truncated hemoglobin from the actinomyces Thermobifida fusca (Tf-trHb) displays a robust peroxidase activity, with optimum at acidic pH values, in experiments with the redox mediator ABTS. However, typical peroxidase substrates, such as phenolic or aromatic amine compounds. appear to be poor substrates for Tf-trHb. In turn, the protein is able to catalyze a unique dehydrogenation reaction of dibenzylbutanolides, suggested intermediates in the biosynthesis of podophyllotoxin, in the presence of hydrogen peroxide. Dibenzylbutanolides with a free 4 ''-hydroxyl group were thus converted into the corresponding 2,7 ''-dehydroderivatives thus setting up the basis for an efficient biotransformation of this important precursor. In particular, Tf-trHb mediated oxidation of trans-2-(4 ''-hydroxy-3 '',5 ''-dimethoxybenzyl)-3-(3',4'-methylenedioxy-7'beta-hydroxybenzyl)butanolide 1 into the corresponding benzylidene-benzoyl-gamma-butyrolactone 2 was obtained at high yield and with excellent selectivity. (C) 2009 Elsevier B.V. All rights reserved

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