Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28

Thongpoo, Preeyanuch; McKee, Lauren S.; Araújo, Ana Catarina; Kongsaeree, Prachumporn T.; Brumer, Harry

doi:10.1016/j.bbagen.2012.11.014

Cited by 27 publications

(21 citation statements)

References 52 publications

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“…The structures, which are similar to that reported for the A. aculeatus GH3 enzyme (AaG; PDB entries 4iib, 4iic, 4iid, 4iie, 4iif, 4iig and 4iih; Suzuki et al, 2013), possess the canonical GH3 three-domain structure with an active centre consistent with the recent assignment by the Brumer group (Thongpoo et al, 2013). All three proteins form dimers in the crystal.…”

Section: Introductionsupporting

confidence: 75%

“…In AfG there are an ethylene glycol (between Arg469 NH1 in both chains) and Superposition of the active sites of the enzymes. The catalytic residues proposed for ExoI (PDB entry 1ex1), Asp491 and Glu285 (Thongpoo et al, 2013), are shown superposed on AfG, AoG and AaG (PDB entry 4iig). The structural figures were all produced using CCP4mg (McNicholas et al, 2011).…”

Section: The Enzyme Dimersmentioning

confidence: 99%

“…Complicating matters further, the sequence diversity in the family, coupled to the observation that the acid/base and nucleophile are derived from different domains, has made it difficult to predict the acid/base residue from sequence alone. In this context, mutagenesis and kinetic studies with substrates of different leavinggroup ability (Thongpoo et al, 2013) have been crucial in assigning the acid/base residue of the Aspergillus GH3 enzymes that are the object of the present study.…”

Section: Introductionmentioning

confidence: 99%

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

Agirre

Ariza

Offen

et al. 2016

Acta Crystallogr D Struct Biol

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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.

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Section: Introductionsupporting

confidence: 75%

Section: The Enzyme Dimersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Agirre

Ariza

Offen

et al. 2016

Acta Crystallogr D Struct Biol

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“…33) After translation to protein, the sequence of P1.2 corresponds to residue 20 of the stop codon of glycoside hydrolase family 3 A. niger β-glucosidase (GenBank: CAB75696) that matched with the LC/MS/MS analysis of P1.2, with only one amino acid difference.…”

Section: Hydrolysis Of Cellulosementioning

confidence: 99%

Purification and characterization of three β-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28

Thongpoo

Srisomsap

Chokchaichamnankit

et al. 2014

Bioscience, Biotechnology, and Biochemistry

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Production and utilization of cellulosic ethanol has been limited, partly due to the difficulty in degradation of cellulosic feedstock. β-Glucosidases convert cellobiose to glucose in the final step of cellulose degradation, but they are inhibited by high concentrations of glucose. Thus, in this study, we have screened, isolated, and characterized three β-glycosidases exhibiting highly glucose-tolerant property from Aspergillus niger ASKU28, namely β-xylosidase (P1.1), β-glucosidase (P1.2), and glucan 1,3-β-glucosidase (P2). Results from kinetic analysis, inhibition study, and hydrolysis of oligosaccharide substrates supported the identification of these enzymes by both LC/MS/MS analysis and nucleotide sequences. Moreover, the highly efficient P1.2 performed better than the commercial β-glucosidase preparation in cellulose saccharification, suggesting its potential applications in the cellulosic ethanol industry. These results shed light on the nature of highly glucose-tolerant β-glucosidase activities in A. niger, whose kinetic properties and identities have not been completely determined in any prior investigations.

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“…As shown in Fig. a, the predicted AmBGL17 shared conserved amino acid residues with other putative β‐glucosidases as well as with a known β‐glucosidase from Thermotoga neapolitana that had its structure predicted by three‐dimensional structure homology modeling (Thongpoo et al ., ). The aspartate within the SDW motif is the conserved catalytic nucleophile in GH3 proteins.…”

Section: Discussionmentioning

confidence: 97%

Discovery of two novel β-glucosidases from an Amazon soil metagenomic library

Bergmann

Costa

Gladden

et al. 2013

FEMS Microbiol Lett

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An Amazon soil microbial community metagenomic fosmid library was functionally screened for β-glucosidase activity. Contig analysis of positive clones revealed the presence of two ORFs encoding novel β-glucosidases, AmBGL17 and AmBGL18, from the GH3 and GH1 families, respectively. Both AmBGL17 and AmBGL18 were functionally identified as β-glucosidases. The enzymatic activity of AmBGL17 was further characterized. AmBGL17 was tested with different substrates and showed highest activity on pNPβG substrate with an optimum temperature of 45 °C and an optimum pH of 6. AmBGL17 showed a Vmax of 116 mM s(-1) and Km of 0.30 ± 0.017 mM. This is the first report of β-glucosidases from an Amazon soil microbial community using a metagenomic approach.

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Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28

Cited by 27 publications

References 52 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

Purification and characterization of three β-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28

Discovery of two novel β-glucosidases from an Amazon soil metagenomic library

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