Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding: insights from crystal structures and MD simulation

Abstract

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-β-d-cellobioside (pNPC), p-nitrophenyl-β-d-lactoside (pNPL) and methylumbelliferyl-β-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-β-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (k_cat ) than TrCel7A, but higher Michaelis-Menten (K_M ) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

Keywords: Cel7; Phanerochaete chrysosporium; Trichoderma reesei; cellulase; fluorescence; ligand binding.

Fig. 1

Substrates and fluorescence titrations. (A) Enzyme kinetics experiments were performed with pNPL, pNPC, oNPC and MUC as substrates. (B) Absorbance spectrum of oNPC (blue) and fluorescence spectrum of TrCel7A wildtype at an excitation wavelength of 295 nm (red). (C) Fluorescence titration of TrCel7A and PcCel7D with oNPC. The concentration of TrCel7A was 0.035 μm (□); 0.1 μm (●) or 0.35 μm (○) and the concentration of PcCel7D was 0.5 μm (■). (D) Displacement titration of TrCel7A with cellobiose at different concentrations of oNPC. The concentration of oNPC was 5 μm (○); 10 μm (●), 20 μm (□) or 40 μm (■).

Fig. 2

Overview of protein structures and substrate binding. (A) Crystal structure of the catalytic domain of TrCel7A (light‐grey) with cellononaose bound (yellow; PDB: 4C4C) and tunnel‐enclosing loops highlighted and labelled in green, superposed with PcCel7D (brown; PDB: 1Z3V). The point of cleavage at the catalytic center is indicated in blue, from which the glucose unit subsites are numbered, with plus‐signs towards the reducing end and minus‐signs towards the non‐reducing end of the sugar polymer. Sidechains are shown of the sugar‐binding tryptophan platforms at subsites −7, −4, −2 and +1, as well as selected residues involved in substrate binding near the catalytic center. Hydrogen bonds are indicated in cyan between Tyr247 and 6OH at subsite −2 and between Thr246 and 6OH at +1. (B) The four new crystal structures presented here, of TrCel7A showing the binding of the ligands in the product subsites +1 to +3 at the active site, and on the outside of the protein, relative to binding of cellononaose. Ligand/protein colours are as follows: pNPC, yellow/light‐yellow (PDB: 4UWT); pNPL, cyan/light‐blue (PDB: 7OC8); lactose, green/light‐green (PDB: 7NYT); oNPC, magenta/pink (PDB: 4V0Z); Cellononaose, light‐grey (PDB: 4C4C). The structure images were created with macpymol [71].

Fig. 3

2f_o‐f_c electron density maps for all the sugar ligands bound in the presented TrCel7A structures, those at the product site (left) and those at the surface (right): pNPC in 4UWT (yellow), lactose and pNPL in 7NYT (white), oNPC in 4V0Z (blue), pNPL in 7OC8 (green). Electron density map contour level 1.0 σ. The structure images were created with macpymol [71].

Fig. 4

Comparison of pNPC, pNPL, oNPC and lactose binding at the product subsites of TrCel7A. (A) The pNPL molecule (green) is bound with the sugar units in the “unprimed” position at subsites +1/+2 and with the nitrophenyl group at subsite +3, whereas lactose (white) binds in the “primed” position. The protein backbone and selected residues of the lactose complex (PDB: 7NYT) are shown in blue colour. (B) An overlay of pNPL (green) and pNPC (yellow) viewed from the catalytic center shows common hydrogen bonds between sugar and protein at subsite +1 (3OH to Glu217 and 6OH to Gln175), and the difference in orientation and interactions for 4OH, with Glu217 for the glucose residue of pNPC, and with Gln175 for the galactose residue of pNPL, respectively. (C) The pNPL (green) and pNPC (yellow) ligands, viewed from the active site exit towards the catalytic center, display very similar binding positions. (D) An overlay of pNPC (yellow) and oNPC (blue) shows the difference in binding of the respective nitrophenyl moieties while the cellobiose units overlap closely. In panels (C) and (D) the protein is shown in semitransparent surface representation and selected amino acid residues as sticks. The structure images were created with macpymol [71].

Fig. 5

The non‐productively bound ligands pNPC (yellow; PDB: 4UWT) and oNPC (blue; PDB: 4V0Z) at subsites +1/+2/+3 in TrCel7A superposed on PcCel7D (brown; PDB: 1Z3V) showing the clash between the nitrophenyl group and Asp336 and close contacts with Tyr 378 and Pro379. The structure images were created with macpymol [71].

Fig. 6

Cluster representations of (A) TrCel7A and (B) PcCel7D protein backbone and ligand structures shown over a 500 ps MD simulation trajectory. The protein backbones are coloured by RMSF (root mean square fluctuation), where red represents the largest fluctuations, and blue represents the lowest fluctuations. The structure images were created with VMD [72].

Fig. 7

Distances between substrate and catalytic amino acids during 1 ns of MD simulations of productive binding at subsites −2/−1/+1 of pNPC, pNPL and oNPC, in TrCel7A (A–D) and in PcCel7D (E–H). The red line shows the shortest distance from the glycosidic oxygen O1 to the nearest O atom of the catalytic acid/base (Glu/E217 in TrCel7A; Glu/E212 in PcCel7D). The blue line shows the shortest distance from the anomeric carbon C1 to the nearest O atom of the catalytic nucleophile (Glu/E212 in TrCel7A; Glu/E207 in PcCel7D).

Fig. 8

Snapshots at 500 ps from MD simulations of productive mode binding of nitrophenyl substrates at subsites −2/−1/+1 in TrCel7A and PcCel7D, superposed with the TrCel7A cellononaose complex PDB: 4C4C (blue). (A) TrCel7A with oNPC pose 1 (cyan) and oNPC pose 2 (magenta). In pose 2 the oNPC is less likely to be hydrolyzed, since the 2‐nitro group appears to obstruct protonation of the glycosidic oxygen by the catalytic acid/base Glu217. (B) PcCel7D with pNPC (yellow) and oNPC pose 2 (green). In the oNPC molecule the glucose residue at subsite −1 has flipped from boat to chair conformation. Also, the oNP ring has flipped around so that the 2‐nitro group is pointing “away,” while it was pointing towards the catalytic center in the starting model. The structure images were created with macpymol [71].

Fig. 9

Crystal packing of protein molecules around the substrate molecules bound at the surface of the TrCel7A enzyme in the crystal structures, viewed along two of the twofold symmetry axes in the crystal. Four substrate molecules bind around the symmetry axes, each making interactions with four surrounding protein molecules. The structure shown is the TrCel7A E212Q/pNPC complex (PDB: 4UWT) with pNPC in space‐filling and protein chains in cartoon representation. Colours are arbitrarily chosen for distinction of individual molecules. The structure images were created with macpymol [71].