Structural insights into trypanosomatid Mnk kinase orthologues (kMnks) suggest altered mechanism in the kinase domain.

Mitogen-activated protein kinase (MAPK) interacting protein kinases (Mnk1 and Mnk2) mediated phosphorylation of the eukaryotic initiation factor eIF4E is an important translation initiation control, in Mnk-mediated oncogenic activity and other disease conditions. Thus, Mnk kinases are an important target for therapy. Trypanosomatids are a class of kinetoplastids, some of which are protozoan parasites and cause diseases in humans. While protein translation initiation is well understood in eukaryotes and prokaryotes, there is a lack of sufficient structural information of this process in trypanosomatids. Here, we report that trypanosomatids have one orthologue of Mnk kinase with low overall sequence homology but high homology in the kinase domain and an additional C-terminal domain containing putative calmodulin binding site(s). We show that while many of the domains and motifs are conserved, homology modeling/structure prediction, docking analysis and molecular dynamics simulation studies suggest that trypanosomatid kMnk kinases, kinase domains are present in DFG-in conformation as opposed to the auto-inhibited DFD-out conformation of un-phosphorylated human Mnk1. Furthermore, we observed that several regulatory features are different in trypanosomatid kMnk kinases. Our study indicates that mechanism and regulation in the kinase domain of trypanosomatid kMnks are likely to be altered, and that they can be important drug targets.

Architecture, domain organization, and functional signatures of trypanosomatid keIF4A1s and Plasmodium peIF4A1s suggest conserved functions.

Initiation of translation is the first of the three obligatory steps required for protein synthesis and is carried out by a large number of protein factors called initiation factors in conjunction with ribosomes. One of the key conserved protein factors in eukaryotes that plays a role in this process is eIF4A, which has three homologues in humans with eIF4A1 being the primary factor playing a role in translation initiation. eIF4As are members of the family of DEAD-box helicases that carry out different biological functions. eIF4A1s are recruited to translation initiation complexes via association with eIF4G and have ATP binding, ATP hydrolysis, RNA binding, and unwinding activities. Plasmodium and trypanosomatids such as Leishmania and Trypanosoma are parasites that cause human disease. While mechanistically the function of eIF4A1s in eukaryotes is wellunderstood, the orthologues peIF4A1s and keIF4A1s in Plasmodium and trypanosomatids are not well-studied. Here, we have used bioinformatics tools and homology modelling/structure prediction to study the motifs and functional signatures of Plasmodium and trypanosomatid peIF4A1s/keIF4A1s. We report a high degree of sequence conservation, structural conservation, and conservation of protein-protein interaction signatures of Plasmodium and trypanosomatid peIF4A1s/keIF4A1s in comparison with human eIF4A1. Thus, in spite of the great divergence in evolution between these parasites and higher eukaryotes, there is remarkable conservation of motifs and functional signatures in Plasmodium and trypanosomatid peIF4A1s/keIF4A1s.

Biochemical characteristics of Myceliophthora thermophila recombinant ß-glucosidase (MtBgl3c) applicable in cellulose bioconversion.

The GH3 ß-glucosidase gene of Myceliophthora thermophila (MtBgl3c) has been cloned and heterologously expressed in E. coli for the first time. This study highlights the important characteristics of recombinant MtBgl3c (rMtBgl3c) which make it a promising candidate in industrial applications. Optimization of the production of rMtBgl3c led to 28,000 U L-1. On purification, it has a molecular mass of ∼100 kDa. It is a broad substrate specific thermostable enzyme that exhibits pH and temperature optima at 5.0 and 55 °C, respectively. The amino acid residues Asp287 and Glu514 act as nucleophile and catalytic acid/base, respectively in the enzyme catalysis. Its low Km value (1.28 mM) indicates a high substrate affinity as compared to those previously reported. The rMtBgl3c displays a synergistic action with the commercial enzyme cocktail in the saccharification of sugarcane bagasse suggesting its utility in the cellulose bioconversion. Tolerance to solvents, detergents as well as glucose make this enzyme applicable in wine, detergent, paper and textile industries too.

Structural and thermostability insights into cellobiohydrolase of a thermophilic mould Myceliophthora thermophila: in-silico studies.

Cellobiohydrolase (CBH) is one of the cellulases with a wide range of industrial applications; it plays a pivotal role in cellulose hydrolysis and thus in biofuel production. The structural and thermostability analysis of a CBHII of the thermophilic mold Myceliophthora thermophila (MtCel6A) had been carried out using various in-silico approaches. The validation of 3 D model by the Ramachandran plot indicated 88.5% amino acid residues in the favoured regions. Docking analysis suggested MtCel6A to display a high affinity towards cellotetraose as compared to other substrates. The enzyme exhibited a high tolerance to the end product, cellobiose. The thermostability evaluation by molecular dynamic simulations and principal component analysis confirmed its tolerance to elevated temperatures. The identified thermolabile regions could be targeted for site-directed mutagenesis in order to ameliorate thermostability further. Our experimental data published earlier confirmed the present findings of in-silico studies. The structural and functional characteristics of MtCel6A highlighted its critical features that make it a useful biocatalyst in several industrial processes.Communicated by Ramaswamy H. Sarma.

Structural aspects of ß-glucosidase of Myceliophthora thermophila (MtBgl3c) by homology modelling and molecular docking.

Cellulases are the enzymes with diverse range of industrial applications. Cellulases degrade cellulose into monomeric glucose units by hydrolysing ß-1,4-glycosidic bonds. There are three components of cellulases: a) endoglucanase, b) exoglucanase and c) ß-glucosidase which act synergistically in cellulose bioconversion. The cellulases are the third largest industrial enzymes with a great potential in bioethanol production. In this investigation, a ß-glucosidase of a thermophilic fungus Myceliophthora thermophila (MtBgl3c) was analysed for its structural characterization using in silico approaches. The protein structure of MtBgl3c is unknown, therefore an attempt has been made to model 3D structure using Modeller 9.23 software. The MtBgl3c protein model generated was validated from Verify 3D and ERRAT scores of 89.37% and 71.25%, respectively derived from SAVES. Using RAMPAGE the Ramachandran plot was generated, which predicted the accuracy of the 3D model with 91.5% amino acid residues in the favored region. The ion binding and N-glycosylation sites were also predicted. The generated model was docked with cellobiose to predict the most favorable binding sites of MtBgl3c. The key amino acid residues involved in cellobiose bonding are Val88, Asp106, Asp287, Tyr255, Arg170, Glu514. The catalytic conserved amino residues of MtBgl3c were identified. The dock score of cellobiose with MtBgl3c is much lower (-6.46 kcal/mol) than that of glucose (-5.61 kcal/mol), suggesting its high affinity for cellobiose. The docking data of MtBgl3c with glucose illustrate its tolerance to glucose. The present study provides insight into structural characteristics of the MtBgl3c which can be further validated by experimental data. Highlights3D structure of ß-glucosidase (MtBgl3c) of Myceliophthora thermophila is being proposed based on computational analysesThe amino acid residues Asp106, Asp287, Tyr255, Arg170 and Glu514 have been identified to play catalytically important role in substrate bindingDocking and interaction of MtBgl3c with cellobiose and glucose has been confirmedDocking analysis of MtBgl3c with glucose suggested its glucose toleranceThe data would be useful in engineering enzymes for attaining higher catalytic efficiencyCommunicated by Ramaswamy H. Sarma.

Recombinant cellobiohydrolase of Myceliophthora thermophila: characterization and applicability in cellulose saccharification.

A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4-12) and temperatures (40-100 °C) with optima at pH 10.0 and 60 °C. It exhibits T1/2 of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The Km, Vmax, kcat and kcat/Km values of the enzyme are 3.2 mg mL-1, 222.2 µmol mg-1 min-1, 2492 s-1 and 778.7 s-1 mg-1 mL-1, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries.

Thermostable cellulose saccharifying microbial enzymes: Characteristics, recent advances and biotechnological applications.

Cellulases play a promising role in the bioconversion of renewable lignocellulosic biomass into fermentable sugars which are subsequently fermented to biofuels and other value-added chemicals. Besides biofuel industries, they are also in huge demand in textile, detergent, and paper and pulp industries. Low titres of cellulase production and processing are the main issues that contribute to high enzyme cost. The success of ethanol-based biorefinery depends on high production titres and the catalytic efficiency of cellulases functional at elevated temperatures with acid/alkali tolerance and the low cost. In view of their wider application in various industrial processes, stable cellulases that are active at elevated temperatures in the acidic-alkaline pH ranges, and organic solvents and salt tolerance would be useful. This review provides a recent update on the advances made in thermostable cellulases. Developments in their sources, characteristics and mechanisms are updated. Various methods such as rational design, directed evolution, synthetic & system biology and immobilization techniques adopted in evolving cellulases with ameliorated thermostability and characteristics are also discussed. The wide range of applications of thermostable cellulases in various industrial sectors is described.

Progress in Ameliorating Beneficial Characteristics of Microbial Cellulases by Genetic Engineering Approaches for Cellulose Saccharification.

Lignocellulosic biomass is a renewable and sustainable energy source. Cellulases are the enzymes that cleave ß-1, 4-glycosidic linkages in cellulose to liberate sugars that can be fermented to ethanol, butanol, and other products. Low enzyme activity and yield, and thermostability are, however, some of the limitations posing hurdles in saccharification of lignocellulosic residues. Recent advancements in synthetic and systems biology have generated immense interest in metabolic and genetic engineering that has led to the development of sustainable technology for saccharification of lignocellulosics in the last couple of decades. There have been several attempts in applying genetic engineering in the production of a repertoire of cellulases at a low cost with a high biomass saccharification. A diverse range of cellulases are produced by different microbes, some of which are being engineered to evolve robust cellulases. This review summarizes various successful genetic engineering strategies employed for improving cellulase kinetics and cellulolytic efficiency.