Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma.
Fungi of the Trichoderma species are valued industrial enzymes in support of the 'zero-waste' technology to convert agro-industrial biomass into valuable products, i.e. nanocellulose (NC). In this study, an in silico approach using substrate docking and molecular dynamic (MD) simulation was used to predict the order of which the multilayers of cellulosic polymers, i.e. lignin, hemicellulose and cellulose in oil palm leaves (OPL) are degraded by fungal enzymes, endocellulase and exocellulase. The study aimed to establish the catalytic tendencies of the enzymes to optimally degrade the cellulosic components of OPL for high yield production of NC. Energy minimized endocellulase and exocellulase models revealed satisfactory scores of PROCHECK (90.0% and 91.2%), Verify3D (97.23% and 98.85%) and ERRAT (95.24% and 91.00%) assessments. Active site prediction by blind docking, COACH meta-server and multiple sequence alignment indicated the catalytic triads for endocellulase and exocellulase were Ser116-His205-Glu249 and Ser382-Arg124-Asp385, respectively. Binding energy of endocellulase docked with hemicellulose (-6.0 kcal mol-1) was the most favourable followed by lignin (-5.6 kcal mol-1) and cellulose (-4.4 kcal mol-1). Exocellulase, contrarily, bonded favorably with lignin (-8.7 kcal mol-1), closely followed by cellulose (-8.5 kcal mol-1) and hemicellulose (-8.4 kcal mol-1). MDs simulations showed that interactions of complexes, endocellulase-hemicellulose and the exocellulase-cellulose being the most stable. Thus, the findings of the study successfully identified the specific actions of sugar-acting enzymes for NC production. Communicated by Ramaswamy H. Sarma.
Literature has shown that oil palm leaves (OPL) can be transformed into nanocellulose (NC) by fungal lignocellulosic enzymes, particularly those produced by the Trichoderma species. However, mechanism of β-glucosidase and xylanase selectivity to degrade lignin, hemicellulose and cellulose in OPL for NC production remains relatively vague. The study aimed to comprehend this aspect by an in silico approach of molecular docking, molecular dynamics (MD) simulation and Molecular-mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis, to compare interactions between the β-glucosidase- and xylanase from Trichoderma asperellum UC1 in complex with each substrate. Molecular docking of the enzyme-substrate complex showed residues Glu165-Asp226-Glu423 and Arg155-Glu210-Ser160 being the likely catalytic residues of β-glucosidase and xylanase, respectively. The binding affinity of β-glucosidase for the substrates are as follows: cellulose (-8.1 kcal mol-1) > lignin (-7.9 kcal mol-1) > hemicellulose (-7.8 kcal mol-1), whereas, xylanase showed a corresponding preference for; hemicellulose (-6.7 kcal mol-1) > cellulose (-5.8 kcal mol-1) > lignin (-5.7 kcal mol-1). Selectivity of both enzymes was reiterated by MD simulations where interactions between β-glucosidase-cellulose and xylanase-hemicellulose were the strongest. Notably low free-binding energy (ΔGbind) of β-glucosidase and xylanase in complex with cellulose (-207.23 +/- 47.13 kJ/mol) and hemicellulose (-131.48 +/- 24.57 kJ/mol) were observed, respectively. The findings thus successfully identified the cellulose component selectivity of the polymer-acting β-glucosidase and xylanase of T. asperellum UC1.Communicated by Ramaswamy H. Sarma.
Bacteria blight is one of the most serious bacterial diseases of rice worldwide. The identification of genetic potential against bacterial blight in the existing rice resources is a prerequisite to develop multigenic resistance to combat the threat of climate change. This investigation was conducted to evaluate alleles variation in 38 Malaysian cultivars using thirteen Simple Sequences Repeats markers and one Sequence Tagged Sites (STS) marker which were reported to be linked with the resistance to bacterial blight. Based on molecular data, a dendrogram was constructed which classified the rice cultivars into seven major clusters at 0.0, 0.28 and 0.3 of similarity coefficient. Cluster 5 was the largest group comprised of ten rice cultivars where multiple genes were identified. However, xa13 could not be detected in the current rice germplasm, whereas xa2 was detected in 25 cultivars. Molecular analysis revealed that Malaysian rice cultivars possess multigenic resistance.
Efficacy of a β-1,4-glucosidase from Trichoderma harzianum T12 (ThBglT12) in disrupting the cell wall of the phytopathogenic fungus M. phaseolina (Macrophomina phaseolina) was studied, as the underlying molecular mechanisms of cell wall recognition remains elusive. In this study, the binding location identified by a consensus of residues predicted by COACH tool, blind docking, and multiple sequence alignment revealed that molecular recognition by ThBglT12 occurred through interactions between the α-1,3-glucan, β-1,3-glucan, β-1,3/1,4-glucan, and chitin components of M. phaseolina, with corresponding binding energies of -7.4, -7.6, -7.5 and -7.8 kcal/mol. The residue consensus verified the participation of Glu172, Tyr304, Trp345, Glu373, Glu430, and Trp431 in the active site pocket of ThBglT12 to bind the ligands, of which Trp345 was the common interacting residue. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), total energy, and minimum distance calculation from molecular dynamics (MD) simulation further confirmed the stability and the closeness of the binding ligands into the ThBglT12 active site pocket. The h-bond occupancy by Glu373 and Trp431 instated the role of the nucleophile for substrate recognition and specificity, crucial for cleaving the β-1,4 linkage. Further investigation showed that the proximity of Glu373 to the anomeric carbon of β-1,3/1,4-glucan (3.5 Å) and chitin (5.5 Å) indicates the nucleophiles' readiness to form enzyme-substrate intermediates. Plus, the neighboring water molecule appeared to be correctly positioned and oriented towards the anomeric carbon to hydrolyze the β-1,3/1,4-glucan and chitin, in less than 4.0 Å. In a nutshell, the study verified that the ThBglT12 is a good alternative fungicide to inhibit the growth of M. phaseolina.Communicated by Ramaswamy H. Sarma.
This study presents the initial structural model of L-haloacid dehalogenase (DehLBHS1) from Bacillus megaterium BHS1, an alkalotolerant bacterium known for its ability to degrade halogenated environmental pollutants. The model provides insights into the structural features of DehLBHS1 and expands our understanding of the enzymatic mechanisms involved in the degradation of these hazardous pollutants. Key amino acid residues (Arg40, Phe59, Asn118, Asn176, and Trp178) in DehLBHS1 were identified to play critical roles in catalysis and molecular recognition of haloalkanoic acid, essential for efficient binding and transformation of haloalkanoic acid molecules. DehLBHS1 was modeled using I-TASSER, yielding a best TM-score of 0.986 and an RMSD of 0.53 Å. Validation of the model using PROCHECK revealed that 89.2% of the residues were located in the most favored region, providing confidence in its structural accuracy. Molecular docking simulations showed that the non-simulated DehLBHS1 preferred 2,2DCP over other substrates, forming one hydrogen bond with Arg40 and exhibiting a minimum energy of -2.5 kJ/mol. The simulated DehLBHS1 exhibited a minimum energy of -4.3 kJ/mol and formed four hydrogen bonds with Arg40, Asn176, Asp9, and Tyr11, further confirming the preference for 2,2DCP. Molecular dynamics simulations supported this preference, based on various metrics, including RMSD, RMSF, gyration, hydrogen bonding, and molecular distance. MM-PBSA calculations showed that the DehLBHS1-2,2-DCP complex had a markedly lower binding energy (-21.363 ± 1.26 kcal/mol) than the DehLBHS1-3CP complex (-14.327 ± 1.738 kcal/mol). This finding has important implications for the substrate specificity and catalytic function of DehLBHS1, particularly in the bioremediation of 2,2-DCP in contaminated alkaline environments. These results provide a detailed view of the molecular interactions between the enzyme and its substrate and may aid in the development of more efficient biocatalytic strategies for the degradation of halogenated compounds.Communicated by Ramaswamy H. Sarma.