rd
23 Congress of the International Union for Biochemistry and Molecular Biology
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44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
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th
Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF FUNGAL ENZYMES
FROM GLYCOSIDE HYDROLASES FAMILY 12: ENDOGLUCANASES VS
XYLOGLUCANASES
Calzado, F.1,3; Prates, E.2; Gonçalves, T.A.1,3; Rubio, M.V.1,3; Zubieta, M.P1,3.;Squina,
F.M.1; Skaf, M.S.2; Damásio, A.R.L.1,4*
1
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional
de Pesquisa em Energia e Materiais (CNPEM), Campinas-SP, Brazil; 2Instituto de
Química, Universidade Estadual de Campinas (UNICAMP), Campinas-SP, Brazil;
3
Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas-SP,
Brazil; 4Instituto de Pesquisa em Bioenergia (IPBEN), Universidade Estadual Paulista
“Julio de Mesquita Filho” (UNESP), Rio Claro-SP, Brazil.
Xyloglucan is the predominant hemicellulose in primary cell walls of dicots and several
monocots. Enzymes that cleave the xyloglucan backbone at unbranched glucose
residues have been identified in glycoside hydrolases (GH) families 5, 7, 12, 16, 44, and
74. In this work three GH12 glucanases from Aspergillus clavatus (AclaXegA), A.
zonatus (AspZo) and A. terreus (AtEglD) were studied. AclaXegA is strictly specific to
xyloglucan while AtEglD and AspZo cleave beta-glucan and xyloglucan. The
phylogenetic tree of Aspergilli GH12 enzymes reveals two well-defined clades
representing endoglucanases and xyloglucanases. AspZo was included in the
xyloglucanases group based on phylogenetic and structural characteristics, as well as
AclaXegA, but based on functional analysis its enzymatic activity was characteristic of
promiscuous endoglucanases such as AtEglD AspZo supposedly represent a link
between two group of enzymes. The main objective of this work was to understand why
some GH12 enzymes are strictly specific for xyloglucan hydrolysis. We characterized
these enzymes by biochemical studies and Molecular Dynamics Simulations (MDS).
The first question was why AclaXegA has no activity on beta-glucan? By MDS we
conclude that the region of AclaXegA that accommodates the substrate reducing-end is
primarily composed by non-polar residues and the unbranched glucose of beta-glucan
does not bind properly. However, AtEglD and AspZo are primarily composed by polar
residues in the equivalent region and properly bind unbranched glucoses. The second
question was what is the key residue for xyloglucan hydrolysis? The residue Y111,
localized at the loop 1 and conserved in all the endoglucanases analyzed, is essential
for the catalysis of xyloglucan by AtEglD. Moreover, the Y111 has equivalent residues in
AspZo (Y30) and AclaXegA (Y19). In summary, AspZo has a deletion in the loop1
(corresponding to Y111 in AtEglD) but has an equivalent residue that determine the
hydrolysis of xyloglucan (Y30), as well as AclaXegA (Y19).
Key words: Xyloglucanase; GH12; Xyloglucan.
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)