The Slowdown of the Endoglucanase <i>Trichoderma reesei</i> Cel5A-Catalyzed Cellulose Hydrolysis Is Related to Its Initial Activity

One important feature of hydrolysis of cellulose by cellulases is that the reaction slows down quickly after it starts. In this work, we investigate the slowdown mechanism at the early stage of the reaction using endoglucanase <i>Tr</i>. Cel5A-catalyzed phosphate acid-swollen cellulose (PASC) hydrolysis as a model system. Specifically, we focus on the effect of enzyme adsorption on the reaction slowdown. Nineteen single mutations are introduced (with the assistance of molecular dynamics simulations) to perturb the enzyme PASC interaction, yielding the adsorption partitioning coefficient <i>K</i>_r that ranged from 0.12 to 0.39 L/g, compared to that of the wild type (0.26 L/g). Several residues, including T18, K26, Y26, H229, and T300, are demonstrated to be important for adsorption of the enzyme to PASC. The kinetic measurements show that the slowdown of the hydrolysis is not correlated with the adsorption quantified by the partitioning coefficient <i>K</i>_r but is anticorrelated with the initial activity. This result suggests that the mutants with higher activity are more prone to being trapped or deplete the most reactive substrate faster and the adsorption plays no apparent role in the reaction slowdown. The initial activity of Cel5A against PASC is correlated with the enzyme specific activity against a soluble substrate <i>p</i>-nitrophenyl cellobioside

Carboxyl–Peptide Plane Stacking Is Important for Stabilization of Buried E305 of Trichoderma reesei Cel5A

Hydrogen bonds or salt bridges are usually formed to stabilize the buried ionizable residues. However, such interactions do not exist for two buried residues D271 and E305 of Trichoderma reesei Cel5A, an endoglucanase. Mutating D271 to alanine or leucine improves the enzyme thermostability quantified by the temperature <i>T</i>₅₀ due to the elimination of the desolvation penalty of the aspartic acid. However, the same mutations for E305 decrease the enzyme thermostability. Free energy calculations based on the molecular dynamics simulation predict the thermostability of D271A, D271L, and E305A (compared to WT) in line with the experimental observation but overestimate the thermostability of E305L. Quantum mechanical calculations suggest that the carboxyl–peptide plane stacking interactions occurring to E305 but not D271 are important for the carboxyl group stabilization. For the protonated carboxyl group, the interaction energy can be as much as about −4 kcal/mol for parallel stacking and about −7 kcal/mol for T-shaped stacking. For the deprotonated carboxyl group, the largest interaction energies for parallel stacking and T-shaped stacking are comparable, about −7 kcal/mol. The solvation effect generally weakens the interaction, especially for the charged system. A search of the carboxyl–peptide plane stacking in the PDB databank indicates that parallel stacking but not T-shaped stacking is quite common, and the most probable distance between the two stacking fragments is close to the value predicted by the QM calculations. This work highlights the potential role of carboxyl amide π–π stacking in the stabilization of aspartic acid and glutamic acid in proteins

Engineering a More Thermostable Blue Light Photo Receptor <i>Bacillus subtilis</i> YtvA LOV Domain by a Computer Aided Rational Design Method

<div><p>The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the <i>T</i>_m of the flavin mononucleotide based fluorescent protein <i>Bacillus Subtilis</i> YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.</p></div

Flowchart of designing thermostable FbFP mutants.

<p>Briefly, FoldX followed by FEC (free energy calculation) are used to search for potential thermostable single mutants, from which a dozen are selected for experimental tests. The distribution of thermostable mutants is analyzed to identify the “hot spot”. Then more mutants in the “hot spot” are calculated by FEC and those predicted to be more stable are tested by experiments. Finally all stabilizing mutants are pooled together and multiple mutants are combined to further improve the protein's stability.</p

Residues in close contact with I120 (A, B), F107 (C, D), F124 (E, F) and F111 (G, H) are labeled where (A, C, E, G) are from subunit 1 and (B, D, F, H) are from subunit 2.

<p>Residues in close contact with I120 (A, B), F107 (C, D), F124 (E, F) and F111 (G, H) are labeled where (A, C, E, G) are from subunit 1 and (B, D, F, H) are from subunit 2.</p

Thermal denaturation of the WT FbFP, the single point mutant N124Y, and the triple mutant N107Y-N124Y-M111F.

<p>The fluorescence intensity of the bound FMN is used to monitor the protein denaturation. As can be seen, the mutants have higher percentages of fluorescence at elevated temperature than WT suggesting mutations increase FbFP thermostability.</p

Melting temperatures of WT and mutant FbFP.

a<p><i>T</i>_m was not determined due to the weak fluorescence of the sample at room temperature.</p

Locations of mutated sites exhibiting improved thermostability.

<p>WT residues of the mutated sites are highlighted in yellow and labeled in red. The two subunits are drawn in grey and dark cyan respectively. Residues H22, V25, N107, D109, M111, V120 and N124 are from the dimer interface. The figure was drawn based on FbFP x-ray structure 2PR5 by using Discovery Studio Visualizer program.</p