Biochemical Characterization and Crystal Structures of a Fungal Family 3 β-Glucosidase, Cel3A from Hypocrea jecorina

Saeid Karkehabadi,Kate E Helmich,Thijs Kaper,Henrik Hansson,Nils-Egil Mikkelsen,Mikael Gudmundsson,Kathleen Piens,Meredith Fujdala,Goutami Banerjee,John S Scott-Craig,Jonathan D Walton,George N Phillips,Mats Sandgren

doi:10.1074/jbc.m114.587766

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.

Highlights

The whole cellulase mixture produced by natural H. jecorina isolates contains about 1% HjCel3A when induced using sophorose [54]
When a portion of the whole cellulase mixture was replaced with purified HjCel3A on an equal protein basis, the conversion of both phosphoric acid-swollen cellulose (PASC) and Pretreated corn stover (PCS) increased (Fig. 1)
These results agree with reports that overexpression of the HjCel3A-encoding gene in H. jecorina improves cellulose and lignocellulosic substrate conversion [9, 15]

Summary

EXPERIMENTAL PROCEDURES

H. jecorina whole cellulase mixture Accellerase௡ 1000 was a kind gift from Genencor. Corn stover was pretreated with dilute sulfuric acid by the United States Department of Energy National Renewable Energy Laboratory, washed, and adjusted to pH 5. Culture filtrate from production of HjCel3A in H. jecorina was diluted 10-fold with 25 mM sodium acetate, pH 4.0 (acetate buffer), and incubated at 37 °C for 30 min. Equal volumes (50 ␮l) of serially diluted HjCel3A or variants were mixed with 15 mM cellobiose in 50 mM sodium acetate, pH 5.0 (final concentration, 7.5 mM cellobiose) in a 96-well microtiter plate. A volume of 190 ␮l of the substrate was added to 10 ␮l of enzyme at an appropriate concentration, and the release of 2-chloro-4-nitrophenol (CNP) was followed spectrophotometrically every 10 min at 37°C by monitoring the absorbance at 405 nm on a Microplate Reader model EL808 (Bio-Tek Instruments, Inc.). Crystallization, Data Collection, Structure Determination, and Refinement—Purified HjCel3A was concentrated to 3.9 mg/ml in a buffer containing 25 mM sodium acetate, pH 4.0, and 100 mM sodium chloride. All figures were created in PyMOL [51]

RESULTS AND DISCUSSION

Data collection and processing

Cellobiose Cellotriose Cellotetraose Gentiobiose Laminaribiose Sophorose CNPG mM