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X-ray structure and function studies of key enzymes for biomass conversion

GH6 cellobiohydrolases and GH61 lytic polysaccharide monooxygenases (LPMO)

Wu, Miao (2013). X-ray structure and function studies of key enzymes for biomass conversion. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis agriculturae Sueciae, 1652-6880 ; 2013:23
ISBN 978-91-576-7785-3
[Doctoral thesis]

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Abstract

The need for large enzyme quantities due to the difficult hydrolysis of recalcitrant polysaccharides is still a major barrier to economical biomass conversion for biofuel production. To discover or develop new efficient wood degrading enzymes and add into enzyme cocktails are essential for optimizing enzymatic conversion of biomass.

Cellulases in glycoside hydrolase family 6 (GH6) play a key role and are obvious targets for enzyme discovery and engineering. However, convenient substrates for high throughput screening have not been available. In paper I, improved fluorogenic substrates for GH6 were rationally designed, synthesized and evaluated. Hydrolysis rates increased by 10–150 times with Hypocrea jecorina Cel6A. Enzyme-substrate structures showed that the modifications led to relief of the exo-anomeric effect and a better position of the glycosidic bond for protonation. In paper II, the first crystal structure of a bacterial GH6 cellobiohydrolase, Thermobifida fusca Cel6B, reveals that the enzymes has a much longer substrate binding tunnel than its fungal GH6 counterparts and that the tunnel exit is closed by a loop that needs to be displaced to allow cellobiose product release for processive action by the enzyme.

Recently, lytic polysaccharide monooxygenases (LPMOs) were discovered as a new class of enzyme for cleavage of recalcitrant polysaccharides with a novel oxidative mechanism. In paper III and IV, one fungal LPMO, Phanerochaete chrysosporium GH61D, was recombinantly expressed and shown to be metal-dependent, to cleave glycosidic bonds in wood lignocellulose and to oxidize preferentially at carbon C1. The PchGH61D crystal structure is the first structure of an LPMO from basidiomycetes. Its active center shows a type II copper binding configuration, which is common to other LPMOs. Structure comparison and molecular dynamic simulations indicate three loops and a series of aromatic and polar residues near the binding surface that may influence substrate recognition and binding.

Authors/Creators:Wu, Miao
Title:X-ray structure and function studies of key enzymes for biomass conversion
Subtitle:GH6 cellobiohydrolases and GH61 lytic polysaccharide monooxygenases (LPMO)
Series/Journal:Acta Universitatis agriculturae Sueciae (1652-6880)
Year of publishing :2013
Volume:2013:23
Number of Pages:58
Papers/manuscripts:
NumberReferences
I.Wu M*, Nerinckx W*, Piens K, Ishida T, Hansson H, Sandgren M, Ståhlberg J. (2013). Rational design, synthesis, evaluation and enzymesubstrate structures of improved fluorogenic substrates for family 6 glycoside hydrolase. FEBS J 280(1), 184-98.
II.Sandgren M, Wu M, Karkehabadi S, Mitchinson C, Kelemen BR, Larenas EA, Ståhlberg J, Hansson H. (2013). The structure of a bacterial cellobiohydrolase: the catalytic core of the Thermobifida fusca family GH6 cellobiohydrolase Cel6B. J Mol Biol 425(3), 622-635.
III.Westereng B, Ishida T, Vaaje-Kolstad G, Wu M, Eijsink VGH, Igarashi K, Samejima M, Ståhlberg J, Horn SJ, Sandgren M. (2011). The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metaldependent oxidative enzyme that cleaves cellulose. PloS One 6(11):e27807
IV.Wu M, Beckham GT, Larsson AM, Ishida T, Kim S, Payne CM, Himmel ME, Crowley MF, Horn SJ, Westereng B, Igarashi K, Samejima M, Ståhlberg J, Eijsink VGH, Sandgren M. (2013). Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the basidiomycota fungus Phanerochaete chrysosporium. J Biol Chem 288, doi: 10.1074/jbc.M113.459396
Place of Publication:Uppsala
Publisher:Dept. of Molecular Biology, Swedish University of Agricultural Sciences
ISBN for printed version:978-91-576-7785-3
ISSN:1652-6880
Language:English
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:X Agricola extesions > X30 Life sciences
X Agricola extesions > X50 Chemistry
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Structural Biology
(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Biochemistry and Molecular Biology
Agrovoc terms:enzymatic hydrolysis, enzymes, hydrolases, phanerochaete chrysosporium, biomass
Keywords:cellobiohydrolase, Lytic polysaccharide monooxygenase, GH6, GH61, LPMO, methylumbelliferyl-β-cellobioside, Phanerochaete chrysosporium, Thermobifida fusca, Hypocrea jecorina
URN:NBN:urn:nbn:se:slu:epsilon-e-1430
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-e-1430
ID Code:10351
Department:(NL, NJ) > Dept. of Molecular Biology (until 131231)
Deposited By: Ph.D Miao Wu
Deposited On:03 May 2013 10:43
Metadata Last Modified:02 Dec 2014 11:00

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