The New Putative Type III Effector SkP48 in Bradyrhizobium sp. DOA9 is Involved in Legume Nodulation

Bradyrhizobium sp. DOA9 can nodulate a wide spectrum of legumes; however, unlike other bradyrhizobia, DOA9 carries a symbiotic plasmid harboring type III secretion system (T3SS) and several effector (T3E) genes, one of which encodes a new putative type III effector—SkP48. Here, we demonstrated the pivotal roles of SkP48 from Bradyrhizobium sp. DOA9 in inhibiting nodulation of various Vigna species and Crotalaria juncea and suppressing nodulation efficiency of Arachis hypogea. By contrast, the nodulation efficiency of a SkP48 mutant did not differ significantly with the DOA9 wild-type strain on Macroptilium atropurpureum and Stylosanthes hamata. An evolutionary analysis revealed that the SkP48 effector which contains a shikimate kinase and a SUMO protease (C48 cysteine peptidase) domain is distinct from the others effectors previously identified in others bradyrhizobia and pathogenic bacteria. Our findings suggest that the new putative T3E SkP48 is a key factor suppressing nodulation and nodule organogenesis in several legumes by activation of effector-triggered immunity through salicylic acid biosynthesis induction, which is deleterious to rhizobial infection. In addition, nodulation may be modulated by the function of defensins involved in jasmonic acid signalling in V. radiata SUT1.

Exploring the Mechanism of Shikimate Kinase through Quantum Mechanical and Molecular Mechanical (QM/MM) Methods

The chemical step of Shikimate Kinase Helicobacter pylori, involving the transfer of a phosphoryl group, has been studied by using quantum mechanical and molecular mechanical (QM/MM) methods. Understanding the mechanism of this chemical step, present in bacteria and other microorganisms but absent in humans, can lead to the development of novel drugs for the treatment of common diseases caused by those pathogenic organisms. Different mechanisms including associative, dissociative, and concerted have been proposed up to now but there is not a consensus on the type of pathway that the reaction follows. Herein, we found that the mechanism has features from the associative and concerted types. An analysis of the free energy landscape of the chemical step reveals that the reaction is a two-step process without a well-defined intermediate state.

Structure-based Discovery of Narirutin as a Shikimate kinase Inhibitor with Anti-tubercular Potency

Background:: Shikimate pathway is essential for tubercular bacillus but it is absent in mammals. Therefore, Shikimate kinase and other enzymes in the pathway are potential targets for the development of novel anti-tuberculosis drugs. Objective:: In the present study, Shikimate kinase is selected as the target for in silico screening of phytochemicals with an aim to discover a novel herbal drug against Mycobacterium tuberculosis (Mtb). Methods:: A structure-based drug discovery approach is undertaken for the execution of the objective. Virtual screening of phytochemical database NPACT against the target, Shikimate kinase (PDB ID 3BAF), is carried out followed by toxicity and drug-likeness filtration. Finally, a lead, narirutin was selected for in vitro anti-tubercular study. Results:: Narirutin, present in citrus fruits, emerges as the lead. It is considered to be non-toxic with predicted high LD50 value, 12000 mg/kg body weight. The phytochemical is tested for its antitubercular activity in vitro. It has MIC99 62.5 μg/mL against the MtbH37Rv strain. Conclusions:: This is the first-ever report to show anti-tuberculosis potency of narirutin.

Novel kinase platform for the validation of the anti-tubercular activities of Pelargonium sidoides (Geraniaceae)

Background Pelargonium sidoides is an important traditional medicine in South Africa with a well-defined history of both traditional and documented use of an aqueous-ethanolic formulation of the roots of P. sidoides (EPs 7630), which is successfully employed for the treatment of respiratory tract infections. There is also historical evidence of use in the treatment of tuberculosis. The aim of this study was to develop a platform of Mycobacterium tuberculosis (Mtb) kinase enzymes that may be used for the identification of therapeutically relevant ethnobotanical extracts that will allow drug target identification, as well as the subsequent isolation of the active compounds. Results Mtb kinases, Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase were cloned, produced in Escherichia coli and characterized. HPLC-based assays were used to determine the enzyme activities and subsequently the inhibitory potentials of varying concentrations of a P. sidoides extract against the produced enzymes. The enzyme activity assays indicated that these enzymes were active at low ATP concentrations. The 50% inhibitory concentration (IC50) of an aqueous root extract of P. sidoides against the kinases indicated SK has an IC50 of 1.2 μg/ml and GK 1.4 μg/ml. These enzyme targets were further assessed for compound identification from the P. sidoides literature. Conclusion This study suggests P. sidoides is potentially a source of anti-tubercular compounds and the Mtb kinase platform has significant potential as a tool for the subsequent screening of P. sidoides extracts and plant extracts in general, for compound identification and elaboration by selected extract target inhibitor profiling.

Virtual Screening, Molecular Docking and Dynamic Simulation of Shikimate Kinase from Mycobacterium Tuberculosis Using in Silico Approach

<p>Shikimate kinase (SK) is an enzyme that catalyzes the fifth steps in the shikimate pathway. The enzyme facilitate the transfer of phosphoryl from ATP to shikimate, to produce ADP and shikimate-3-phosphate from <i>Mycobacterium tuberculosis</i> (MTB). The 3D structure of SK bound ligands (4-(2-Hydroxyethyl)-1-Piperazine Ethanesulfonic Acid (EPE)), ADP and metals (Mg2+, Cl- and Pt+) obtained from PDB (PDB ID: 1L4U and resolution 1.8Å). The structural analysis of the SK revealed that it has a substrate or shikimate binding site (Asp34, Arg58, and Lys136) and substrate binding via amide nitrogen (Gly80). It also possessed nucleotide binding region (Gly12─Thr17), the ATP binding site (Arg117 and Arg153) and metallic ion (Mg2+) binding site (Ser16 and Asp32). All these residues mentioned above play an essential role in the catalytic activity of the SK. Therefore inhibition any of these residues serve as a stumbling block for the normal function of the enzyme. A total of eleven thousand three hundred and twenty-three (11323) compounds obtained from two public databases (Zinc Database and PubChem) capable of binding to SK with good binding affinities. These compounds further filtered for Lipinski’s rule of five, drug-likeness, molecular docking analysis, and ADME and toxicity analysis. Three compounds with minimum binding energies─ PubChem15478 (─11.75 kcal/mol), ZINC02838601 (─11.52 kcal/mol), and ZINC11790367 (─9.88 kcal/mol) ─were selected and used for the MD simulation analysis. Also, MD simulation of the SK bound to EPE, ADP, and Mg2+ were carried out to compare their stabilities with the selected protein-ligand complexes. The result showed that the two compounds (ZINC11790367 and PubChem15478) formed stable and rigid complexes comparable to the bound ligand and the cofactors during the 50ns MD simulation. Therefore, it concluded that the above mentioned two compounds capable of inhibiting SK considered as prospective drugs for MTB after successful experimental validation.</p>

Virtual Screening, Molecular Docking and Dynamic Simulation of Shikimate Kinase from Mycobacterium Tuberculosis Using in Silico Approach

<p>Shikimate kinase (SK) is an enzyme that catalyzes the fifth steps in the shikimate pathway. The enzyme facilitate the transfer of phosphoryl from ATP to shikimate, to produce ADP and shikimate-3-phosphate from <i>Mycobacterium tuberculosis</i> (MTB). The 3D structure of SK bound ligands (4-(2-Hydroxyethyl)-1-Piperazine Ethanesulfonic Acid (EPE)), ADP and metals (Mg2+, Cl- and Pt+) obtained from PDB (PDB ID: 1L4U and resolution 1.8Å). The structural analysis of the SK revealed that it has a substrate or shikimate binding site (Asp34, Arg58, and Lys136) and substrate binding via amide nitrogen (Gly80). It also possessed nucleotide binding region (Gly12─Thr17), the ATP binding site (Arg117 and Arg153) and metallic ion (Mg2+) binding site (Ser16 and Asp32). All these residues mentioned above play an essential role in the catalytic activity of the SK. Therefore inhibition any of these residues serve as a stumbling block for the normal function of the enzyme. A total of eleven thousand three hundred and twenty-three (11323) compounds obtained from two public databases (Zinc Database and PubChem) capable of binding to SK with good binding affinities. These compounds further filtered for Lipinski’s rule of five, drug-likeness, molecular docking analysis, and ADME and toxicity analysis. Three compounds with minimum binding energies─ PubChem15478 (─11.75 kcal/mol), ZINC02838601 (─11.52 kcal/mol), and ZINC11790367 (─9.88 kcal/mol) ─were selected and used for the MD simulation analysis. Also, MD simulation of the SK bound to EPE, ADP, and Mg2+ were carried out to compare their stabilities with the selected protein-ligand complexes. The result showed that the two compounds (ZINC11790367 and PubChem15478) formed stable and rigid complexes comparable to the bound ligand and the cofactors during the 50ns MD simulation. Therefore, it concluded that the above mentioned two compounds capable of inhibiting SK considered as prospective drugs for MTB after successful experimental validation.</p>