Modulating the thermostability of Endoglucanase I from Trichoderma reesei using computational approaches.

In the last decades, effective cellulose degradation became a major point of interest due to the properties of cellulose as a renewable energy source and the widespread application of cellulases (the cellulose degrading enzymes) in many industrial processes. Effective bioconversion of lignocellulosic biomass into soluble sugars for ethanol production requires use of thermostable and highly active cellulases. The library of current cellulases includes enzymes that can work at acidic and neutral pH in a wide temperature range. However, only few cellulases are reported to be thermostable. In order to alleviate this, we have performed a hybrid approach for the thermostabilization of a key cellulase, Endoglucanase I (EGI) from Trichoderma reesei. We combined in silico and in vitro experiments to modulate the thermostability of EGI. Four different predictive algorithms were used to set up a library of mutations. Three thermostabilizer mutations (Q126F, K272F, Q274V) were selected and molecular dynamics simulations at room temperature and high temperatures were performed to analyze the effect of the mutations on enzyme structure and stability. The mutations were then introduced into the endoglucanase 1 gene, using site-directed mutagenesis, and the effect of the mutations on enzyme structure and stability were determined. MD simulations supported the fact that Q126F, K272F and Q274V mutations have a thermostabilizing effect on the protein structure. Experimental studies validated that all of the mutants exhibited higher thermostability compared with native EGI albeit with a decrease in specific activity.

Modifying the catalytic preference of tributyrin in Bacillus thermocatenulatus lipase through in-silico modeling of enzyme-substrate complex.

In this study, rational design for Bacillus thermocatenulatus lipase (BTL2) was carried out to lower the activation barrier for hydrolysis of short-chain substrates. In this design, we used computational models for the enzyme-substrate (ES) complexes of tributyrin (C4) and tricaprylin (C8), which were generated through docking and molecular dynamics (MD) simulations. These ES complexes were employed in steered MD (SMD) simulations with Jarzynski's equality to estimate their relative binding free energies. Potential mutation sites for modifying the chain-length selectivity of BTL2 were found by inspecting the SMD trajectories and fine-tuning the volume and hydrophobicity of the cleft. Seven mutations (F17A, L57F, V175A, V175F, I320A, I320F and L360F) were performed to cover three binding pockets for sn-1, sn-2 and sn-3 acyl chains. The relative binding free energies of the mutant ES complexes formed by C4 and C8 ligands were calculated similarly. The experimental routines of protein engineering including site-directed mutagenesis, heterologous protein expression and purification were performed for all lipases. Steady-state specific activities towards C4 and C8 were determined for wild-type and mutant lipases, which gave an estimate of the relative change in the binding free energy of transition state complex (ES(‡)). The chain-length selectivity of mutants was determined from the relative changes in the activation barrier of hydrolysis of C4 and C8 triacylglycerol with respect to wild-type using computational and experimental findings. The most promising mutant for C4 over C8 preference was found to be L360F. We suggest that L360F may be at a critical position to lower the activation barrier for C4 and elevate it for C8 hydrolysis.

From in silico to in vitro: modelling and production of Trichoderma reesei endoglucanase 1 and its mutant in Pichia pastoris.

In this study, a major cellulase, namely endoglucanase 1 (EGI) from Trichoderma reesei was mutated by the introduction of four different lysine and glycine rich loops to create a hotspot for directed crosslinking of EGI away from the active site. The impact of the inserted loops on the stability of the enzyme was analyzed using molecular dynamics (MD) and the effect on the active site was studied using molecular mechanics (MM) simulations. The best loop mutation predicted in silico (EGI_L5) was introduced to EGI via site directed mutagenesis. The loop mutant EGI_L5 and EGI were both expressed in Pichia pastoris. Enzymes were characterized and their activities against soluble substrates such as CMC and 4-MUC were determined. Both enzymes exhibited similar pH and temperature activity and thermal stability profiles. Moreover, specific activity of EGI_L5 against 4-MUC was found to be the same as the native enzyme.

Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis.

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.

Clustering of protein families into functional subtypes using Relative Complexity Measure with reduced amino acid alphabets.

BACKGROUND: Phylogenetic analysis can be used to divide a protein family into subfamilies in the absence of experimental information. Most phylogenetic analysis methods utilize multiple alignment of sequences and are based on an evolutionary model. However, multiple alignment is not an automated procedure and requires human intervention to maintain alignment integrity and to produce phylogenies consistent with the functional splits in underlying sequences. To address this problem, we propose to use the alignment-free Relative Complexity Measure (RCM) combined with reduced amino acid alphabets to cluster protein families into functional subtypes purely on sequence criteria. Comparison with an alignment-based approach was also carried out to test the quality of the clustering. RESULTS: We demonstrate the robustness of RCM with reduced alphabets in clustering of protein sequences into families in a simulated dataset and seven well-characterized protein datasets. On protein datasets, crotonases, mandelate racemases, nucleotidyl cyclases and glycoside hydrolase family 2 were clustered into subfamilies with 100% accuracy whereas acyl transferase domains, haloacid dehalogenases, and vicinal oxygen chelates could be assigned to subfamilies with 97.2%, 96.9% and 92.2% accuracies, respectively. CONCLUSIONS: The overall combination of methods in this paper is useful for clustering protein families into subtypes based on solely protein sequence information. The method is also flexible and computationally fast because it does not require multiple alignment of sequences.

Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling.

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.