Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus<i>Humicola grisea</i>var.<i>thermoidea</i>

Momeni, Majid Haddad; Goedegebuur, Frits; Hansson, Henrik; Karkehabadi, Saeid; Askarieh, Glareh; Mitchinson, Colin; Larenas, E.; Ståhlberg, Jerry; Sandgren, Mats

doi:10.1107/s1399004714013844

Cited by 25 publications

(27 citation statements)

References 55 publications

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“…Regarding the former, Grassick et al () reported that the B2 loop of Re Cel7A (in which the current mutations are made) was flexible to the extent that the loop was not visible in the electron density map and this obviously speak against strong A3–B2 loop interactions in the unligated form. A high degree of fluctuation of the B2 loop has also been reported in Hg Cel7A (Momeni et al, ) and Hi Cel7A (Momeni et al, ). One possible joint interpretation of this structural and kinetic information is that the B2 loop engages in interactions with the substrate and the A3 loop late in the association path, when the transition state of this process has been passed and the cellulose strand is gradually filling the binding tunnel.…”

Section: Discussionmentioning

confidence: 60%

“…Conversely, Cel7D from Phanerochaete chrysosporium ( Pc Cel7D), which is also a processive cellobiohydrolase, has a number of loop deletions and hence much more accessible substrate binding region (Munoz et al, ). Other fungal Cel7As, including enzymes from Hypocrea hazianum ( Hz Cel7A) (Textor et al, ), Humicola grisea ( Hg Cel7A) (Momeni et al, ), and Heterobasidion irregulare ( Hi Cel7A) (Momeni et al, ) have intermediate degrees of tunnel coverage and loop fluctuations.…”

Section: Introductionmentioning

confidence: 99%

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Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose

Sørensen

Windahl

McBrayer

et al. 2016

Biotech & Bioengineering

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Cel7A cellobiohydrolases perform processive hydrolysis on one strand of cellulose, which is threaded through the enzyme's substrate binding tunnel. The tunnel structure results from a groove in the catalytic domain, which is covered by a number of loops. These loops have been identified as potential targets for engineering of this industrially important enzyme family, but only few systematic studies on this have been made. Here we show that two asparagine residues (N194 and N197) positioned in the loop covering the glucopyranose subsite -4 (recently denoted B2 loop) of the thermostable Cel7A from Rasamsonia emersonii had profound effects on both substrate interactions and catalytic efficacy. At room temperature the double mutant N194A/N197A showed strongly reduced substrate affinity with a water-cellulose partitioning coefficient threefold lower than the wild type. Yet, this variant was catalytically efficient with a maximal turnover about twice as high as the wild type. Analogous but smaller changes were found for the single mutants. Analysis of these changes in affinity and kinetics as a function of temperature, led to the conclusion that replacement of N194 and particularly N197 with alanine leads to faster enzyme-substrate dissociation. Conversely, these residues appeared to have little or no effect on the rate of association. We suggest that the controlled adjustment of the enzyme-substrate dissociation prompts faster cellulolytic enzymes. Biotechnol. Bioeng. 2017;114: 53-62. © 2016 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose

Sørensen

Windahl

McBrayer

et al. 2016

Biotech & Bioengineering

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“…RemCel7A, Melanocarpus albomyces Cel7B, Humicola grisea var. thermoidea Cel7A, and A. fumigatus Cel7A [22,24,49,50]. As a result, the GcaCel7A ligand fluctuates slightly more than that of HjeCel7A in the À7 and À6 sites (Fig.…”

Section: Discussionmentioning

confidence: 95%

“…Three-dimensional structures of the catalytic domains of nine GH7 CBHs have been published previously, the first being HjeCel7A and the most recent being Aspergillus fumigatus Cel7A [19][20][21][22][23][24][25][26][27]. The structures share a common b-jelly roll fold with a curved b-sandwich constructed from two largely antiparallel b-sheets packing face-to-face, forming an approximately 50-A-long substrate-binding groove along the GH7 catalytic module.…”

Section: Introductionmentioning

confidence: 99%

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C

et al. 2015

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The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulosebinding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50°C versus 60-65°C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported -on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A.

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“…The crystal structure of CBH-1 from a number of filamentous fungi shows an N-terminal pGlu modification (20, 21). The crystal structures of additional GH7 members, including EG1 (Cel7b) (22, 23) and Cel7D (24) have an N-terminal pGlu.…”

Section: Resultsmentioning

confidence: 99%

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

Dana

Iavarone

et al. 2017

mBio

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The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.

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Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungusHumicola griseavar.thermoidea

Cited by 25 publications

References 55 publications

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

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