Potential of MurA Enzyme and GBAP in Fsr Quorum Sensing System as Antibacterial Drugs Target: In vitro and In silico Study of Antibacterial Compounds from Myrmecodia pendans.

BACKGROUND: Increasing the resistance issue has become the reason for the development of new antibacterial in crucial condition. Many ways are tracked to determine the most effective antibacterial agent. Some proteins that are a key role in bacteria metabolism are targeted, including MurA in cell wall biosynthesis and gelatinase biosynthesis-activating pheromone (GBAP) in Fsr Quorum Sensing (QS) system. OBJECTIVE: The objective of this research is the analysis of compounds 1-4 from M. pendans as antibacterial and anti-QS activity trough protein inhibition by in silico study; focus on the structure-activity relationships, to appraise their role as an antibacterial and anti-QS agent in the molecular level. METHODS: Both activities of M. pendans compounds (1-4) were analyzed by in silico, compared to Fosfomycin, Ambuic acid, Quercetin, and Taxifolin as a standard. Chemical structures of M. pendans compounds were converted using an online program molview. The compounds were docked to MurA, GBAP, gelatinase and serine protease using Autodock Vina in Pyrx 0.8 followed PYMOL to visualization and proteis.plus program to analyze of the complex. RESULTS: All compounds from M. pendans bound on MurA, GBAP, gelatinase and serine protease except compound 2. This biflavonoid did not attach to MurA and serine protease yet is the favorable ligand for GBAP and gelatinase with the binding affinity of -6.9 and -9.4 Kcal/mol respectively. Meanwhile, for MurA and serine protease, compound 4 is the highest of bonding energy with values of -8.7 and -6.4 Kcal/mol before quercetin (MurA, -8.9 Kcal/mol) and taxifolin (serine protease, -6.6 Kcal/mol). CONCLUSION: Based on the data, biflavonoid acts better as anti-QS than an inhibitor of MurA enzyme while the others can be acted into both of them either the therapeutic agent of anti-QS or antibacterial agent of MurA inhibitor.

Cloning, characterization, and enzymatic identification of a new tryptophan decarboxylase from Ophiorrhiza pumila.

Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal-5'-phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT-producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536 bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01 kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa. Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high-efficiency expression of OpTDC2 in Escherichia coli, we fused the TF tag onto the N-terminal of the OpTDC2. Optimum enzymatic activity was observed at 45 °C, pH 8 and cofactor concentration of 0.1 mM. The catalytic reaction was strongly inhibited by metal ions of Cu2+ , Zn2+ , and Fe2+ . The l-tryptophan was particularly catalyzed compared with d-tryptophan. Besides, the Km and kcat of the OpTDC2 were 1.08 mM and 0.78 Sec-1 , respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila.

Physiological, Biochemical, and Transcriptomic Responses of Neolamarckia cadamba to Aluminum Stress.

Aluminum is the most abundant metal of the Earth's crust accounting for 7% of its mass, and release of toxic Al3+ in acid soils restricts plant growth. Neolamarckia cadamba, a fast-growing tree, only grows in tropical regions with acidic soils. In this study, N. cadamba was treated with high concentrations of aluminum under acidic condition (pH 4.5) to study its physiological, biochemical, and molecular response mechanisms against high aluminum stress. High aluminum concentration resulted in significant inhibition of root growth with time in N. cadamba. The concentration of Al3+ ions in the root tip increased significantly and the distribution of absorbed Al3+ was observed in the root tip after Al stress. Meanwhile, the concentration of Ca, Mg, Mn, and Fe was significantly decreased, but P concentration increased. Aluminum stress increased activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase from micrococcus lysodeiktic (CAT), and peroxidase (POD) in the root tip, while the content of MDA was decreased. Transcriptome analysis showed 37,478 differential expression genes (DEGs) and 4096 GOs terms significantly associated with treatments. The expression of genes regulating aluminum transport and abscisic acid synthesis was significantly upregulated; however, the genes involved in auxin synthesis were downregulated. Of note, the transcripts of several key enzymes affecting lignin monomer synthesis in phenylalanine pathway were upregulated. Our results shed light on the physiological and molecular mechanisms of aluminum stress tolerance in N. cadamba.

Genome assembly of Chiococca alba uncovers key enzymes involved in the biosynthesis of unusual terpenoids.

Chiococca alba (L.) Hitchc. (snowberry), a member of the Rubiaceae, has been used as a folk remedy for a range of health issues including inflammation and rheumatism and produces a wealth of specialized metabolites including terpenes, alkaloids, and flavonoids. We generated a 558 Mb draft genome assembly for snowberry which encodes 28,707 high-confidence genes. Comparative analyses with other angiosperm genomes revealed enrichment in snowberry of lineage-specific genes involved in specialized metabolism. Synteny between snowberry and Coffea canephora Pierre ex A. Froehner (coffee) was evident, including the chromosomal region encoding caffeine biosynthesis in coffee, albeit syntelogs of N-methyltransferase were absent in snowberry. A total of 27 putative terpene synthase genes were identified, including 10 that encode diterpene synthases. Functional validation of a subset of putative terpene synthases revealed that combinations of diterpene synthases yielded access to products of both general and specialized metabolism. Specifically, we identified plausible intermediates in the biosynthesis of merilactone and ribenone, structurally unique antimicrobial diterpene natural products. Access to the C. alba genome will enable additional characterization of biosynthetic pathways responsible for health-promoting compounds in this medicinal species.

Suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila.

Camptothecin is a monoterpenoid indole alkaloid that exhibits anti-tumor activity. In Ophiorrhiza pumila, production of camptothecin and its related alkaloids was high in the hairy roots, but not in the cell suspension culture derived from hairy roots. To identify the intermediates in camptothecin biosynthesis, expression of genes encoding tryptophan decarboxylase (TDC) and secologanin synthase (SLS), the two enzymes catalyzing the early steps in camptothecin biosynthesis, were suppressed in the hairy roots of O. pumila by RNA interference (RNAi), and metabolite changes were investigated. In most TDC- and SLS-suppressed lines, accumulation of camptothecin and related alkaloids, strictosidine, strictosamide, pumiloside, and deoxypumiloside was reduced. The accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS. Some hairy root-specific chromatographic peaks detected by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) exhibited positive or negative correlation with TDC expression, suggesting their possible involvement in camptothecin biosynthesis.

Molecular cloning of an O-methyltransferase from adventitious roots of Carapichea ipecacuanha.

Carapichea ipecacuanha produces various emetine-type alkaloids, known as ipecac alkaloids, which have long been used as expectorants, emetics, and amebicides. In this study, we isolated an O-methyltransferase cDNA from this medicinal plant. The encoded protein (CiOMT1) showed 98% sequence identity to IpeOMT2, which catalyzes the 7'-O-methylation of 7'-O-demethylcephaeline to form cephaeline at the penultimate step of emetine biosynthesis (Nomura and Kutchan, J. Biol. Chem., 285, 7722-7738 (2010)). Recombinant CiOMT1 showed both 7'-O-methylation and 6'-O-methylation activities at the last two steps of emetine biosynthesis. This indicates that small differences in amino acid residues are responsible for distinct regional methylation specificities between IpeOMT2 and CiOMT1, and that CiOMT1 might contribute to two sequential O-methylation steps from 7'-O-demethylcephaeline to emetine.

Molecular characterization and expression analysis of a new cDNA encoding strictosidine synthase from Ophiorrhiza japonica.

A new full-length cDNA encoding strictosidine synthase (designated as OjSTR, GenBank Accession No. 1087598), which catalyzes a committed step in camptothecin biosynthetic pathway, was isolated from young leaves of Ophiorrhiza japonica for the first time. OjSTR was 1,258 bp and contained a 1,062 bp open reading frame encoding a deduced protein of 353 amino acid residues. Sequence analyses showed that OjSTR had high homology with other STRs from some TIA-producing plants. Phylogenetic tree analysis showed that OjSTR had closest relationship with STR from O. pumila. Tissue expression pattern analysis revealed that OjSTR constitutively expressed in all the tested tissues at different levels, which was high in flower, moderate in leaf and root, low in stem. Expression profiles under plant defense signals such as methyl jasmonate and salicylic acid were investigated, and the results revealed that expression of OjSTR was all induced, implying that OjSTR was high elicitor responsive.

Genetic variability of the narrow endemic tree Antirhea aromatica Castillo-Campos Lorence, (Rubiaceae, Guettardeae) in a tropical forest of Mexico.

BACKGROUND AND AIMS: Genetic structure and variability were examined in the only three extant populations of the narrow-endemic tree Antirhea aromatica (Rubiaceae, Guettardeae), an endangered species of the tropical forest of eastern Mexico. Patterns of genetic diversity within and among populations for adult plants and seedlings were obtained. METHODS: Allozyme electrophoresis of 15 loci was conducted and the data analysed with statistical approximation for obtaining genetic diversity, structure and gene flow. KEY RESULTS: The mean expected heterozygosity (He) in the adult and seedling populations was 0.18 +/- 0.08 and 0.20 +/- 0.09, respectively. The genetic variation explained by differences among populations was 51 and 35 %, for adult and seedling populations, respectively. On average, gene flow between paired adult populations was low (Nm = 0.26 +/- 0.09), compared with other trees from the tropical forest. CONCLUSIONS: The results indicated that the populations evaluated have high genetic variability, compared with other endemic and geographically narrowly distributed plant species, in areas with high levels of environmental heterogeneity (e.g. tropical forests). The conservation implications of the results are discussed, and in this regard it is proposed that A. aromatica should be considered as an indicator species with economic potential. It is suggested that sustainable management practices should be implemented and that the areas where the species is distributed should be declared a natural reserve to ensure the species conservation.

Chemical and enzymatic hydrolysis of anthraquinone glycosides from madder roots.

For the production of a commercially useful dye extract from madder, the glycoside ruberythric acid has to be hydrolysed to the aglycone alizarin which is the main dye component. An intrinsic problem is the simultaneous hydrolysis of the glycoside lucidin primeveroside to the unwanted mutagenic aglycone lucidin. Madder root was treated with strong acid, strong base or enzymes to convert ruberythric acid into alizarin and the anthraquinone compositions of the suspensions were analysed by HPLC. A cheap and easy method to hydrolyse ruberythric acid in madder root to alizarin without the formation of lucidin turned out to be the stirring of dried madder roots in water at room temperature for 90 min: this gave a suspension containing pseudopurpurin, munjistin, alizarin and nordamnacanthal. Native enzymes are responsible for the hydrolysis, after which lucidin is converted to nordamnacanthal by an endogenous oxidase.

Nucleotide sequence diversity at the methionine synthase locus in endangered Dunnia sinensis (Rubiaceae): an evaluation of the positive selection hypothesis.

Methionine synthase is a key enzyme for the synthesis of the aspartate-derived methionine, the immediate precursor of S-adenosyl-methionine, which has been illustrated to be associated with plant growth and pathogen interactions. In this study we tested the positive selection hypothesis of molecular evolution of the methionine synthase gene in Dunnia sinensis. In the entire sample of 87 sequences, 22 haplotypes of introns and 16 haplotypes of exons were identified. An excess of polymorphism over the neutral expectation for the class of unique nucleotide polymorphisms was observed in both exon and intron sequences. Ten replacement substitutions versus six synonymous substitutions among lineages, although nonsignificant, revealed that some advantageous mutants might have been favored. The distribution of d(N)/d(S) > 1 at nodes between closely related haplotypes in the gene network also indicated weak and variable positive selection. Nevertheless, low levels of genetic diversity in exons (theta; = 0.0052) and introns (theta; = 0.0070) of the methionine synthase gene of the outcrossing Dunnia were also attributed to the endangered status of the species. The atpB-rbcL intergenic spacer of cpDNA and the ribosomal internal transcribed spacer of mtDNA were used to discern the relative effectiveness of natural selection from intrinsic evolutionary forces. The low levels of nucleotide polymorphisms in both organelle spacers and the significant population differentiation reflected the effect of population-species history and demography. Two major lineages of the methionine synthase gene genealogy were recovered corresponding to two geographic regions, a result that was consistent with organelle phylogenies. Both past fragmentation and recent habitat disturbance causing complete bottlenecks may have resulted in population decline and geographic isolation and may have led to the depletion of genetic variation at loci in nuclear and organelle genomes.