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High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with alpha-L-arabinofuranosidase activity
KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
KTH, School of Biotechnology (BIO), Industrial Biotechnology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden .
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2015 (English)In: Acta crystallographica. Section F, Structural biology communications, Vol. 71, no Pt 3, 338-45 p.Article 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
2015. Vol. 71, no Pt 3, 338-45 p.
National Category
Natural Sciences
URN: urn:nbn:se:kth:diva-164102DOI: 10.1107/S2053230X15003337ISI: 000351157900016PubMedID: 25760712ScopusID: 2-s2.0-84924655292OAI: diva2:803841
Swedish Research Council Formas, 2013-1741Swedish Research Council, 2013-5717

QC 20150419

Available from: 2015-04-13 Created: 2015-04-13 Last updated: 2015-04-29Bibliographically approved
In thesis
1. Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
Open this publication in new window or tab >>Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.

In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).

To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.

In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from  H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of  HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.

Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 57 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:8
se conversion, galacto-oligosaccharides, thermostability, propeptide
National Category
Structural Biology
Research subject
urn:nbn:se:kth:diva-165613 (URN)978-91-7595-511-7 (ISBN)
Public defence
2015-05-26, FB55, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)

QC 20150429

Available from: 2015-04-29 Created: 2015-04-29 Last updated: 2015-04-29Bibliographically approved

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