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Structural insights into the catalytic mechanism, protein dynamics, inhibition and thermostability of GH7 cellobiohydrolases

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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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
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
Volume:2014:32
Number of Pages:58
Papers/manuscripts:
NumberReferences
I.Momeni MH*, Payne CM*, Hansson H, Mikkelsen NE, Svedberg J, Engström Å, Sandgren M, Bechkham GT and Ståhlberg J. (2013). Structural, Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare. J Biol Chem 288, 5861-5872.
II.Knott BC, Momeni MH, Crowley MF, Mackenzie LF, Götz AW, Sandgren M, Withers SG, Ståhlberg J and Beckham GT (2013). The mechanism of cellulose hydrolysis by a two-step, retaining cellobiohydrolase elucidated by structural and transition path sampling studies. J Am Chem Soc 136, 321-329.
III.Momeni MH, Goedegebuur F, Hansson H, Karkehabadi S, Askarieh G, Mitchinson, C, Larenas E, Ståhlberg J, Sandgren M. Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea. (Submitted to Acta cryst D)
IV.Momeni MH, Ubhayasekera W, Hansson H, Sandgren M., Stahlberg J. Structural insights into the inhibition of Cellobiohydrolase Cel7A by xylooligosaccharides. (Manuscript)
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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