Momeni, Majid Haddad
(2014).
Structural insights into the catalytic mechanism, protein dynamics, inhibition and thermostability of GH7 cellobiohydrolases.
Diss. (sammanfattning/summary)
Uppsala :
Sveriges lantbruksuniv.,
Acta Universitatis Agriculturae Sueciae, 1652-6880
; 2014:32
ISBN 978-91-576-8012-9
eISBN 978-91-576-8013-6
[Doctoral thesis]
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PDF
19MB |
Abstract
Glycoside hydrolase family 7 cellobiohydrolases (GH7 CBH) are typically the most abundant enzymes of cellulolytic fungi and play a key role in biomass recycling in Nature, as well as in biofuel production from plant biomass. This thesis examines molecular properties of this biologically and industrially important class of enzymes.
Paper I shows that HirCel7A is the most abundant protein of the serious forest pathogen Heterobasidion irregulare. The HirCel7A exhibits intermediate dynamical and structural properties between CBHs with the most closed and most open tunnels known in GH7. The results point to tunnel-enclosing loops as important for carbohydrate processivity and association-dissociation on cellulose. Paper II presents the first Michaelis complex, with cellononaose spanning 42 Å of the active site, and the first glycosyl-enzyme intermediate trapped in a GH7 CBH. QM/MM calculations determine optimal reaction coordinates, and rate constants at 11 s⁻¹ for Step1 and 5300 s⁻¹ for Step2, showing that the glycosylation step is rate-limiting. A product-assisted mechanism is revealed for the deglycosylation step, indicating that expulsion of the cellobiose product is not required prior to hydrolysis of the intermediate.
In Paper III, HgrCel7A from Humicola grisea var. thermoidea showed 10 °C higher Tm and 75% higher yield in a biomass performance assay at 65 °C than the canonical HjeCel7A of Hypocrea jecorina. The crystal structure of HgrCel7A indicates higher flexibility in tunnel-defining loops, and structural features potentially related to thermostability and enhanced activity, including a putative conformational switch in an active-center loop not reported previously in GH7. In Paper IV, structures of HjeCel7A in complex with xylooligosaccharides of DP 3-5 show predominant binding in the beginning of the tunnel and partial occupancy for a second binding mode near the catalytic centre. Birchwood xylan displayed ~100-fold stronger inhibition based on mass, suggesting that it may penetrate further into the tunnel and occupy a longer stretch of the active site.
Authors/Creators: | Momeni, Majid Haddad | ||||||||||
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Title: | Structural insights into the catalytic mechanism, protein dynamics, inhibition and thermostability of GH7 cellobiohydrolases | ||||||||||
Series/Journal: | Acta Universitatis Agriculturae Sueciae (1652-6880) | ||||||||||
Year of publishing : | April 2014 | ||||||||||
Number: | 2014:32 | ||||||||||
Number of Pages: | 58 | ||||||||||
Papers/manuscripts: |
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Place of Publication: | Uppsala | ||||||||||
Publisher: | Dept. of Chemistry and Biotechnology, Swedish University of Agricultural Sciences | ||||||||||
ISBN for printed version: | 978-91-576-8012-9 | ||||||||||
ISBN for electronic version: | 978-91-576-8013-6 | ||||||||||
ISSN: | 1652-6880 | ||||||||||
Language: | English | ||||||||||
Publication Type: | Doctoral thesis | ||||||||||
Full Text Status: | Public | ||||||||||
Agris subject categories.: | P Natural resources > P06 Renewable energy resources X Agricola extesions > X30 Life sciences | ||||||||||
Subjects: | (A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Structural Biology | ||||||||||
Agrovoc terms: | cellulase, hydrolases, catalytic activity, stability | ||||||||||
Keywords: | Cellulase, Cellobiohydrolases, Retaining mechanism | ||||||||||
URN:NBN: | urn:nbn:se:slu:epsilon-e-1863 | ||||||||||
Permanent URL: | http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-e-1863 | ||||||||||
ID Code: | 11132 | ||||||||||
Faculty: | NL - Faculty of Natural Resources and Agricultural Sciences (until 2013) | ||||||||||
Department: | (NL, NJ) > Department of Chemistry and Biotechnology (140101-161231) | ||||||||||
External funders: | FORMAS | ||||||||||
Deposited By: | Student Majid Haddad Momeni | ||||||||||
Deposited On: | 16 Apr 2014 14:01 | ||||||||||
Metadata Last Modified: | 14 Dec 2014 17:02 |
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