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

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

doi:10.1074/jbc.m114.587766

Cited by 74 publications

(85 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The domain organization in the crystal structures of AfG and AoG is very similar to those observed in X-ray structures of the GH family 3 (GH3) -d-glucosidases TnBgl3B, AaG, KmBgl1 (from Kluyveromyces marxianus; PDB entry 3abz; Yoshida et al, 2010) and HjCel3A (from Hypocrea jecorina; PDB entry 3zyz; Karkehabadi et al, 2014). In contrast, a smallangle X-ray scattering (SAXS) dummy-atom model calculated for the A. niger -d-glucosidase from GH3 (AnBgl1) reveals a more linear molecular arrangement (Lima et al, 2013), in which the FnIII domain is located away from domains A and B on an extension provided by the linker peptide.…”

Section: Resultssupporting

confidence: 60%

Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases

Agirre

Ariza

Offen

et al. 2016

Acta Crystallogr D Struct Biol

View full text Add to dashboard Cite

The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo-and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant -d-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. -d-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 -d-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley -d-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as highmannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the d-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy 4 C 1 chair conformation and should be of general application to refinements of other crystal structures containing O-or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal -d-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.

show abstract

Section: Resultssupporting

confidence: 60%

Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases

Agirre

Ariza

Offen

et al. 2016

Acta Crystallogr D Struct Biol

View full text Add to dashboard Cite

show abstract

“…Among them, cel3A (bgl1) is the major extracellular β-glucosidase in cellulase production [5], while cel1A (bgl2) [13] and cel1B [14] are intracellular. In addition, it is presumed that cel3B, cel3E, cel3F, cel3G and cel3H are extracellular, while cel3C, cel3D and cel3H are intracellular [15].…”

Section: Introductionmentioning

confidence: 99%

A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production

Lin

et al. 2016

Microb Cell Fact

View full text Add to dashboard Cite

BackgroundThe conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although β-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear.ResultsThe T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular β-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed.ConclusionsThe T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular β-glucosidase hyper production, high cellulase production within a shorter time and a better resistance to carbon catabolite repression. Disruption of β-glucosidase cel3D in TRB1 was identified, which might contribute to the superiority of TRB1 over RUT-C30 and might play a role in the cellulase production. These results laid a foundation for future investigations to further improve cellulase enzymatic efficiency and reduce cost for T. reesei cellulase production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0550-3) contains supplementary material, which is available to authorized users.

show abstract

“…To clarify interaction mode of glucose and ␤-glucosidase, MOE 2010 program was used to simulate the molecular docking for better understanding of the stabilizing effect of glucose. The selected docked conformation owned the best free energy of binding and was closed to a Hypocrea ␤-glucosidase published in PDB database [25] with similar values of docking potential energy (data not listed), indicating our simulation result was reasonable. The model exhibited the 11 active amino residues in catalytic pocket which were responsible for glucose to protect the enzyme from thermo-denaturation were totally overlapped by the catalytic section for cellobiose hydrolysis ( Fig.…”

Section: Molecular Dockingmentioning

confidence: 67%

“…Importantly, the intrinsic fluorescence emission spectra results that heat treated enzyme in the presence of glucose occurred less red shift than that without glucose showed glucose contributed to conformational stabilization of the whole enzyme molecule against thermal denaturation. Structural analysis mentioned that one ␤-glucosidase molecule combined with one glucose molecule in active center [25]. Glucose molecules could form hydrogen bonds to combine with cellulase in active center as glycoside substrate does.…”

Section: Molecular Dockingmentioning

confidence: 99%

Heat inactivation kinetics of Hypocrea orientalis β-glucosidase with enhanced thermal stability by glucose

Shi

et al. 2015

International Journal of Biological Macromolecules

View full text Add to dashboard Cite

Thermal inactivation kinetics of Hypocrea orientalis β-glucosidase and effect of glucose on thermostability of the enzyme have been determined in this paper. Kinetic studies showed that the thermal inactivation was irreversible and first-order reaction. The microscopic rate constants for inactivation of free enzyme and substrate-enzyme complex were both determined, which suggested that substrates can protect β-glucosidase against thermal deactivation effectively. On the other hand, glucose was found to protect β-glucosidase from heat inactivation to remain almost whole activity below 70°C at 20mM concentration, whereas the apparent inactivation rate of BG decreased to be 0.3×10(-3)s(-1) in the presence of 5mM glucose, smaller than that of sugar-free enzyme (1.91×10(-3)s(-1)). The intrinsic fluorescence spectra results showed that glucose also had stabilizing effect on the conformation of BG against thermal denaturation. Docking simulation depicted the interaction mode between glucose and active residues of the enzyme to produce stabilizing effect.

show abstract

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

Cited by 74 publications

References 67 publications

Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases

Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases

A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production

Heat inactivation kinetics of Hypocrea orientalis β-glucosidase with enhanced thermal stability by glucose

Contact Info

Product

Resources

About