A knowledge-driven protocol for prediction of proteins of interest with an emphasis on biosynthetic pathways.

This protocol describes a stepwise process to identify proteins of interest from a query proteome derived from NGS data. We implemented this protocol on Moringa oleifera transcriptome to identify proteins involved in secondary metabolite and vitamin biosynthesis and ion transport. This knowledge-driven protocol identifies proteins using an integrated approach involving sensitive sequence search and evolutionary relationships. We make use of functionally important residues (FIR) specific for the query protein family identified through its homologous sequences and literature. We screen protein hits based on the clustering with true homologues through phylogenetic tree reconstruction complemented with the FIR mapping. The protocol was validated for the protein hits through qRT-PCR and transcriptome quantification. Our protocol demonstrated a higher specificity as compared to other methods, particularly in distinguishing cross-family hits. This protocol was effective in transcriptome data analysis of M. oleifera as described in Pasha et al.•Knowledge-driven protocol to identify secondary metabolite synthesizing protein in a highly specific manner.•Use of functionally important residues for screening of true hits.•Beneficial for metabolite pathway reconstruction in any (species, metagenomics) NGS data.

Homology Modeling Identifies Crucial Amino-Acid Residues That Confer Higher Na+ Transport Capacity of OcHKT1;5 from Oryza coarctata Roxb.

HKT1;5 loci/alleles are important determinants of crop salinity tolerance. HKT1;5s encode plasmalemma-localized Na+ transporters, which move xylem Na+ into xylem parenchyma cells, reducing shoot Na+ accumulation. Allelic variation in rice OsHKT1;5 sequence in specific landraces (Nona Bokra OsHKT1;5-NB/Nipponbare OsHKT1;5-Ni) correlates with variation in salt tolerance. Oryza coarctata, a halophytic wild rice, grows in fluctuating salinity at the seawater-estuarine interface in Indian and Bangladeshi coastal regions. The distinct transport characteristics of the shoots and roots expressing the O. coarctata OcHKT1;5 transporter are reported vis-à-vis OsHKT1;5-Ni. Yeast sodium extrusion-deficient cells expressing OcHKT1;5 are sensitive to increasing Na+ (10-100 mM). Electrophysiological measurements in Xenopus oocytes expressing O. coarctata or rice HKT1;5 transporters indicate that OcHKT1;5, like OsHKT1;5-Ni, is a Na+-selective transporter, but displays 16-fold lower affinity for Na+ and 3.5-fold higher maximal conductance than OsHKT1;5-Ni. For Na+ concentrations >10 mM, OcHKT1;5 conductance is higher than that of OsHKT1;5-Ni, indicating the potential of OcHKT1;5 for increasing domesticated rice salt tolerance. Homology modeling/simulation suggests that four key amino-acid changes in OcHKT1;5 (in loops on the extracellular side; E239K, G207R, G214R, L363V) account for its lower affinity and higher Na+ conductance vis-à-vis OsHKT1;5-Ni. Of these, E239K in OcHKT1;5 confers lower affinity for Na+ transport, as evidenced by Na+ transport assays of reciprocal site-directed mutants for both transporters (OcHKT1;5-K239E, OsHKT1;5-Ni-E270K) in Xenopus oocytes. Both transporters have likely analogous roles in xylem sap desalinization, and differences in xylem sap Na+ concentrations in both species are attributed to differences in Na+ transport affinity/conductance between the transporters.

Dataset for the combined transcriptome assembly of M. oleifera and functional annotation.

In this paper, we present the data acquired during transcriptome analysis of the plant Moringa oleifera [1] from five different tissues (root, stem, leaf, flower and seed) by RNA sequencing. A total of 271 million reads were assembled with an N50 of 2094 bp. The combined transcriptome was assessed for transcript abundance across five tissues. The protein coding genes identified from the transcripts were annotated and used for orthology analysis. Further, enzymes involved in the biosynthesis of select medicinally important secondary metabolites, vitamins and ion transporters were identified and their expression levels across tissues were examined. The data generated by RNA sequencing has been deposited to NCBI public repository under the accession number PRJNA394193 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA394193).

The transcriptome enables the identification of candidate genes behind medicinal value of Drumstick tree (Moringa oleifera).

Moringa oleifera is a plant well-known for its nutrition value, drought resistance and medicinal properties. cDNA libraries from five different tissues (leaf, root, stem, seed and flower) of M. oleifera cultivar Bhagya were generated and sequenced. We developed a bioinformatics pipeline to assemble transcriptome, along with the previously published M. oleifera genome, to predict 17,148 gene models. Few candidate genes related to biosynthesis of secondary metabolites, vitamins and ion transporters were identified. Expressions were further confirmed by real-time quantitative PCR experiments for few promising leads. Quantitative estimation of metabolites, as well as elemental analysis, was also carried out to support our observations. Enzymes in the biosynthesis of vitamins and metabolites like quercetin and kaempferol are highly expressed in leaves, flowers and seeds. The expression of iron transporters and calcium storage proteins were observed in root and leaves. In general, leaves retain the highest amount of small molecules of interest.

Three-dimensional Modelling of the Voltage-gated Sodium Ion Channel from Anopheles gambiae Reveals Spatial Clustering of Evolutionarily Conserved Acidic Residues at the Extracellular Sites.

BACKGROUND: The eukaryotic voltage-gated sodium channel(e-Nav) is a large asymmetric transmembrane protein with important functions concerning neurological function. No structure has been resolved at high resolution for this protein. METHODS: A homology model of the transmembrane and extracellular regions of an Anopheles gambiae para-like channel with emphasis on the pore entrance has been constructed, based upon the templates provided by a prokaryotic sodium channel and a potassium two-pore channel. The latter provides a template for the extracellular regions, which are located above the entrance to the pore, which is likely to open at a side of a dome formed by these loops. RESULTS: A model created with this arrangement shows a structure similar to low-resolution cryoelectron microscope images of a related structure. The pore entrance also shows favorable electrostatic interface. CONCLUSION: Residues responsible for the negative charge around the pore have been traced in phylogeny to highlight their importance. This model is intended for the study of pore-blocking toxins.

Functional relevance of dynamic properties of Dimeric NADP-dependent Isocitrate Dehydrogenases.

BACKGROUND: Isocitrate Dehydrogenases (IDHs) are important enzymes present in all living cells. Three subfamilies of functionally dimeric IDHs (subfamilies I, II, III) are known. Subfamily I are well-studied bacterial IDHs, like that of Escherischia coli. Subfamily II has predominantly eukaryotic members, but it also has several bacterial members, many being pathogens or endosymbionts. subfamily III IDHs are NAD-dependent. The eukaryotic-like subfamily II IDH from pathogenic bacteria such as Mycobacterium tuberculosis IDH1 are expected to have regulation similar to that of bacteria which use the glyoxylate bypass to survive starvation. Yet they are structurally different from IDHs of subfamily I, such as the E. coli IDH. RESULTS: We have used phylogeny, structural comparisons and molecular dynamics simulations to highlight the similarity and differences between NADP-dependent dimeric IDHs with an emphasis on regulation. Our phylogenetic study indicates that an additional subfamily (IV) may also be present. Variation in sequence and structure in an aligned region may indicate functional importance concerning regulation in bacterial subfamily I IDHs. Correlation in movement of prominent loops seen from molecular dynamics may explain the adaptability and diversity of the predominantly eukaryotic subfamily II IDHs. CONCLUSION: This study discusses possible regulatory mechanisms operating in various IDHs and implications for regulation of eukaryotic-like bacterial IDHs such as that of M. tuberculosis, which may provide avenues for intervention in disease.

Determination of phosphorylation sites for NADP-specific isocitrate dehydrogenase from mycobacterium tuberculosis.

Isocitrate Dehydrogenase (ICD) catalyzes the oxidative decarboxylation reaction of 2R,3S-isocitrate to yield 2-oxoglutarate in the Tricarboxylic Acid (TCA) cycle. Two isoforms of NADP-specific ICDs with the E.C number 1.1.1.42 have been annotated in the organism Mycobacterium tuberculosis, monomeric ICD2 and dimeric ICD1. BLAST search against the Protein Data Bank (PDB) database shows a marked similarity between dimeric Mycobacterium tuberculosis ICD1 sequence and that of Sus scrofa, a cytosolic eukaryotic ICD (65% identity). Escherischia coli ICD shows less sequence similarity than the eukaryotic structure. A Homology model has thus been built for M. tuberculosis ICD1 using Sus scrofa and human ICD as templates. Inactivation of ICD1 by phosphorylation similar to E. coli ICD is important to open up the shunt pathway in the TCA cycle, which has been indicated in the case of M. tuberculosis. We therefore attempted to identify a number of likely phosphorylation sites in M. tuberculosis using pattern prediction and checked with the homology models for the accessibility of the peptides containing Serine. It was found that the homologous Serine by alignment with E. coli on M. tuberculosis ICD1 is difficult to access by specific kinases. Hence other probable sites of phosphorylation were checked and three highly probable serine-containing peptides were identified. The effect of phosphorylation at each of these sites was determined by checking the degree of conformational changes, the differences caused by the effect of phosphorylation in the active-site and other apparent motion different from that of the control, i.e., unphosphorylated M. tuberculosis ICD1 model, using molecular dynamics simulations.