Structural, Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare*

Momeni, Majid Haddad; Payne, Christina M.; Hansson, Henrik; Mikkelsen, N.E.; Svedberg, Jesper; Engström, Åke; Sandgren, Mats; Beckham, Gregg T.; Ståhlberg, Jerry

doi:10.1074/jbc.m112.440891

Cited by 68 publications

(130 citation statements)

References 61 publications

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“…As shown, the HjCel7A loop does not open during the 250-ns MD simulation (44), whereas the LqCel7B exo loop undergoes a conformational change between a closed and open state quite readily on the nanosecond time scale. This difference in exo-loop behavior is likely to increase the propensity of LqCel7B to engage in endotype initiation on the cellulose surface relative to HjCel7A (35), as suggested in previous studies of P. chrysosporium Cel7D and HjCel7A (35,44). Lastly, we note that there are significant protein fluctuations at the product site of LqCel7B not observed in HjCel7A.…”

Section: Resultsmentioning

confidence: 99%

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Kern

McGeehan

Streeter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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103

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Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.gribble | carbohydrate degrading enzymes

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Section: Resultsmentioning

confidence: 99%

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Kern

McGeehan

Streeter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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103

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“…In Serratia marcescens chitinases, we found that ligand fluctuations and solvation as well as fluctuation of the localized catalytic residues correlated well with previously measured values of apparent processivity between endochitinases and chitobiohydrolases [47]*. In our comparison of the Heterobasidion irregulare Cel7A structure to T. reesei Cel7A and P. chrysosporium Cel7D, we discovered that active site loop regions of the more processive T. reesei Cel7A open and close at a significantly lower rate than the less processive P. chrysosporium Cel7D and putatively less processive H. irregulare Cel7A suggesting the latter two enzymes may perform endo-initiation events more often than T. reesei Cel7A, which can affect processive ability [52]. Each of these studies qualitatively highlighted the potential contributions of enzyme dynamics to processive ability.…”

Section: Thermodynamics Of Cbh Processivitymentioning

confidence: 87%

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Beckham

Ståhlberg

Knott

et al. 2014

Current Opinion in Biotechnology

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Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases (GHs), which abstract single carbohydrate chains from polymer crystals and cleave glycosidic linkages without dissociating after each catalytic event.Understanding the molecular-level features and structural aspects of processivity is of importance due to the prevalence of processive GHs in biomass-degrading enzyme cocktails. Here, we describe recent advances towards the development of a molecular-level theory of processivity for cellulolytic and chitinolytic enzymes, including the development of novel methods for measuring rates of key steps in processive action and insights gained from structural and computational studies. Overall, we present a framework for developing structure-function relationships in processive GHs and outline additional progress towards developing a fundamental understanding of these industrially important enzymes. IntroductionStructural polysaccharides, such as cellulose and chitin, typically arrange in insoluble, polymeric crystals that form significant components of plant, fungal, and algal cell walls. Microorganisms have evolved suites of enzymatic machinery to degrade these polysaccharides to soluble units for food and energy. These enzyme cocktails are primarily composed of various glycoside hydrolases (GHs) with synergistic functions to efficiently cleave the glycosidic linkages [1,2]. More recently, additional enzymatic functions beyond the canonical GH enzyme battery have been discovered including oxidative enzymes that selectively cleave glycosidic bonds [3][4][5][6][7]. GH cocktails contain enzymes typically delineated into two broadly defined classes: cellobiohydrolases (CBHs) and endoglucanases (EGs) for cellulose depolymerization, or chitobiohydrolases and endochitinases for chitin depolymerization. EGs are thought to randomly hydrolyze glycosidic linkages primarily in amorphous regions of polymer fibers. Alternatively, CBHs are able to attach to carbohydrate chains and processively hydrolyze disaccharide units from the end of a chain without dissociation after each catalytic event. Processivity is traditionally thought to be a means of conserving energy during enzymatic function, and is a general strategy used in the synthesis, modification, and depolymerization of many natural biopolymers [8]. It is this ability to act processively that imparts significant hydrolytic potential to CBHs from various GH families such as GH Family 6, 7, 18, and 48 and typically makes them the most abundant enzymes in natural secretomes of many microorganisms. Thus, GHs are the focus of intense protein engineering efforts for the biofuels industry [9*,10].

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“…At the same time, the side chain of Asp345 in loop B4 of DdiCel7A and DpuCel7A is suitably positioned for H bonding to OH1 of the glucosyl unit at position ϩ2 of the reducing end. Actually, most GH7 CBHs have aspartate at the corresponding position, but TreCel7A and other Trichoderma species lack this interaction due to a 1-residue deletion in loop B4 (15,50,65).…”

Section: Resultsmentioning

confidence: 99%

“…Glutamine at this position is a rare motif among GH7 CBHs and is found only in another protist, Pseudotrichonympha grassii, a parabasilian symbiont in the gut of termites. A few distantly related basidiomycetes have glutamate at this position; e.g., in HirCel7A, molecular dynamics simulations showed that the glutamate side chain can interact with a bound cellulose chain (15). DpuCel7A and DdiCel7A also possess on the A1 loop two glutamine residues (Q97 and Q99) that would be in direct contact with the cellulose surface when the enzyme is adsorbed (64); TreCel7A possesses only the former of these.…”

Section: Resultsmentioning

confidence: 99%

“…The structures of GH7 catalytic domains are known from 10 CBHs, Trichoderma reesei Cel7A (TreCel7A) (12)(13)(14), Heterobasidion irregulare Cel7A (HirCel7A) (15), Phanerochaete chrysosporium Cel7D (PchCel7D) (16), Talaromyces emersonii Cel7A (RemCel7A) (17), Trichoderma harzianum Cel7A (ThaCel7A) (18), Melanocarpus albomyces Cel7B (MalCel7B) (19), Aspergillus fumigatus Cel7A (AfuCel7A) (20), Humicola grisea var. thermoidea Cel7A (HgtCel7A) (21) Limnoria quadripunctata Cel7B (LquCel7B) (22), and Geotrichum candidum Cel7A (23), and three endoglucanases (EGs), T. reesei Cel7B (24), Humicola insolens Cel7B (25), and Fusarium oxysporum Cel7B (26).…”

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confidence: 99%

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Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

Hobdey

Knott

Momeni

et al. 2016

Appl Environ Microbiol

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Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A CBM and DpuCel7A CBM , which were recombinantly expressed in T. reesei. DdiCel7A CBM and DpuCel7A CBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K i of cellobiose was significantly higher for DdiCel7A CBM and DpuCel7A CBM than for TreCel7A: 205, 130, and 29 M, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life. IMPORTANCE GH7CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungus T. reesei when tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate. Dictyostelia are a class of social amoebae (historically known as slime molds) containing four different groups of terrestrial bacterivores from the kingdom Amoebozoa. During vegetative growth, dictyostelia exist as single-celled organisms; upon starvation, a lack of nutrients becomes preventive for vegetative growth and the cells aggregate into a multicellular slug. Slugs have a defined posterior and anterior, have the ability to migrate, are sensitive to light and temperature, and exhibit an innate immune system. When conditions are sufficiently severe, the slug can form a fruiting body, w...

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Structural, Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare*

Cited by 68 publications

References 61 publications

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

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