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

Abstract

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.

Comparative insights into the saccharification potentials of a relatively unexplored but robust Penicillium funiculosum glycoside hydrolase 7 cellobiohydrolase

“…Most GH7 CBHs from filamentous fungi are multi-modular in nature, consisting of a carbohydrate-binding module (CBM), an O -glycosylated linker, and a large catalytic domain (CD) containing a tunnel for threading cellulose chain [13, 14]. The tunnel-bearing structures allow the enzyme to slide along the cellulose chain to the next cleavage site as the product is released [2].…”

“…Long loops extend the edges of the β-sandwich and form a long substrate-binding groove along the entire GH7 catalytic module. [3, 14–17]. …”

“…The majority of reported differences, however, were observed with length and sequence of loops around substrate-binding paths, catalytic centers or products binding sites [3, 14–16, 18]. Till date, GH7 CBH from Trichoderma reesei (TrCBH1) exhibits the most extensively enclosed tunnel among known GH7 CBH structures, while Phanerochaete chrysosporium Cel7D (PchCel7D) displays the most open active site due to several loop deletions and residue size reductions on the tips of tunnel-enclosing loops [15].…”

“…Simulations of the enzymes in each representative state enabled us to directly examine the effects of substrate and products on protein dynamics. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability which will in turn significantly aid in the commercialization of biofuel processes based on enzymatic depolymerization of polysaccharides [3, 14–16, 18]. …”

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

“…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). All GH7s share a ␤-jelly roll fold, with two antiparallel ␤-sheets packing face to face to form a curved ␤-sandwich.…”