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J Biol Chem. 2014 Nov 7;289(45):31624-37. doi: 10.1074/jbc.M114.587766. Epub 2014 Aug 27.

Biochemical characterization and crystal structures of a fungal family 3 β-glucosidase, Cel3A from Hypocrea jecorina.

Author information

1
From the Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden.
2
the Department of Energy Great Lakes Bioenergy Research Center and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
3
DuPont Industrial Biosciences, Palo Alto, California 94304.
4
the Department of Energy Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan 48824, and.
5
the Department of Energy Great Lakes Bioenergy Research Center and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, the Department of Biochemistry and Cell Biology and Department of Chemistry, Rice University, Houston, Texas 77251 georgep@rice.edu.
6
From the Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden, mats.sandgren@slu.se.

Abstract

Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)-β-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A). Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 β-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn(208) and Asn(310). In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms.

KEYWORDS:

Biofuel; Biomass Conversion; Crystal Structure; Enzyme Structure; Glycoside Hydrolase; Hypocrea jecorina; beta-Glucosidase

PMID:
25164811
PMCID:
PMC4223358
DOI:
10.1074/jbc.M114.587766
[Indexed for MEDLINE]
Free PMC Article

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