Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors

Abstract

The cellobiohydrolase Pc_Cel7D is the major cellulase produced by the white‐rot fungus Phanerochaete chrysosporium, constituting ≈10% of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycoside hydrolases and, like other family members, catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. Previous work described the apo structure of the enzyme. Here we investigate the binding of the product, cellobiose, and several inhibitors, i.e. lactose, cellobioimidazole, Tris/HCl, calcium and a thio‐linked substrate analogue, methyl 4‐S‐β‐cellobiosyl‐4‐thio‐β‐cellobioside (GG‐S‐GG). The three disaccharides bind in the glucosyl‐binding subsites +1 and +2, close to the exit of the cellulose‐binding tunnel/cleft. Pc_Cel7D binds to lactose more strongly than cellobiose, while the opposite is true for the homologous Trichoderma reesei cellobiohydrolase Tr_Cel7A. Although both sugars bind Pc_Cel7D in a similar fashion, the different preferences can be explained by varying interactions with nearby loops. Cellobioimidazole is bound at a slightly different position, displaced ≈2 Å toward the catalytic centre. Thus the Pc_Cel7D complexes provide evidence for two binding modes of the reducing‐end cellobiosyl moiety; this conclusion is confirmed by comparison with other available structures. The combined results suggest that hydrolysis of the glycosyl‐enzyme intermediate may not require the prior release of the cellobiose product from the enzyme. Further, the structure obtained in the presence of both GG‐S‐GG and cellobiose revealed electron density for Tris at the catalytic centre. Inhibition experiments confirm that both Tris and calcium are effective inhibitors at the conditions used for crystallization.

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

“…The overall fold is homologous to other GH7 cellobiohydrolases, comprising a globular and equatorially elongated protein, but with several interesting features particularly in the surface loop regions (Fig. 2B) (33,(38)(39)(40)(41). The active site tunnel, formed by four pairs of curved antiparallel β-sheets packed face to face, extends from the −7 binding site at Trp57 to the +2 binding site near Phe362, representing a distance of ∼48 Å between the tunnel entrance and exit.…”

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

“…All eight glucosyl residues are in the chair conformation, and an intact chain would likely feature a glucosyl residue in subsite -3 also exhibiting a chair conformation. The product binding sites (+1/+2) are occupied in the so-called 'Slide' mode in this structure [63]. This conformation is not active for hydrolysis, as the distance from the -1 anomeric carbon to the nucleophile is 7.1 Å.…”

“…This catalytically-active complex may resemble the theoretical model of the Michaelis complex of T. reesei Cel7A (PDB code 8CEL, Figure 2, bottom right). Immediately following the first catalytic step (Glycosylation), the cellobiose product resides in what we refer to as the 'Unprimed glycosyl-enzyme intermediate' (GEI) mode (seen in PDB structures 6CEL, 7CEL, 1Q2E, 1Z3W, 2RFY, 4HAP, 4IPM, and exemplified by the 7CEL structure in Figure 2, lower left frame) [21,44,63,69,70]. As with the Michaelis complex, no GEI crystal structure has been published to date for a GH Family 7 member.…”

“…Nevertheless, various GH7 crystal structures reveal the existence of two distinct product binding modes [63]. The 'Primed GEI' mode (which essentially overlays 'Slide' mode in the +1/+2 subsites) results from a slight shift in the cellobiose product from the 'Unprimed GEI' mode towards the tunnel exit (seen in PDB structures 3CEL, 1Z3T, 1Z3V, and exemplified by the 3CEL structure in Figure 2, bottom left) [63,71]; this movement creates space for a water molecule (the nucleophile of the Deglycosylation step) to move into the active site in between the two chemical steps [63]. The 'priming' thus refers to the preparation of the cellobiose product and the nucleophilic water for the Deglycosylation reaction.…”

Crystallization of Cellobiohydrolase in the Presence of Cellulose-Degraded Cellobiose: Analysis of Intermolecular Interactions and Association Dynamics

“…Crystallization in the presence of foreign materials has also been studied using the seeding technique to obtain crystals [18][19][20]. Representative structural analysis for binding between endoglucanase (AaCel9A) [19] and cellobiose or cellobiohydrolase (Pc_Cel7D) [20] and cellobiose showed that for Aa-Cel9A and Pc_Cel7D, cellobiose binds in three glucosyl-binding subsites (−1, −2, and +1) and two glucosyl-binding subsites (+1 and +2), respectively. Binding sites in Pc_Cel7D are close to the exit of the cellulose-binding cleft.…”