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Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
KTH, School of Biotechnology (BIO), Glycoscience.
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2013 (English)In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 3, 496-504 p.Article 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 In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit.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.

Place, publisher, year, edition, pages
2013. Vol. 3, 496-504 p.
Keyword [en]
Crystal structure, Lignin degradation, Oligomerization, Propeptide, Pyranose 2-oxidase, Thermostability
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-139900DOI: 10.1016/j.fob.2013.10.010ISI: 000339569800078ScopusID: 2-s2.0-84888118395OAI: diva2:687798
Swedish Research Council, 2008-4045 2011-5768

QC 20140115

Available from: 2014-01-15 Created: 2014-01-15 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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