The Chemical Diversity of Lantana camara: Analyses of Essential Oil Samples from Cuba,Nepal, and Yemen

The aerial parts of Lantana camara L. were collected from three different geographical locations: Artemisa (Cuba), Biratnagar (Nepal), and Sana'a (Yemen). The essential oils were obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry. A cluster analysis of 39 L. camara essential oil compositions revealed eight major chemotypes: β‐caryophyllene, germacrene D, ar‐curcumene/zingiberene, γ‐curcumen‐15‐al/epi‐β‐bisabolol, (E)‐nerolidol, davanone, eugenol/alloaromadendrene, and carvone. The sample from Cuba falls into the group dominated by (E)‐nerolidol, the sample from Nepal is a davanone chemotype, and the sample from Yemen belongs to the β‐caryophyllene chemotype. The chemical composition of L. camara oil plays a role in the biological activity; the β‐caryophyllene and (E)‐nerolidol chemotypes showed antimicrobial and cytotoxic activities.     

What can genome-scale metabolic network reconstructions do for prokaryotic systematics?

It has recently been proposed that in addition to Nomenclature, Classification and Identification, Comprehending Microbial Diversity may be considered as the fourth tenet of microbial systematics [Staley JT (2010) The Bulletin of BISMiS, 1(1): 1–5]. As this fourth goal implies a fundamental understanding of microbial speciation, this perspective article argues that translation of bacterial genome sequences into metabolic features may contribute to the development of modern polyphasic taxonomic approaches. Genome-scale metabolic network reconstructions (GSMRs), which are the result of computationally predicted and experimentally confirmed stoichiometric matrices incorporating all enzyme and metabolite components encoded by a genome sequence, provide a platform that can illustrate bacterial speciation. As the topology and the composition of GSMRs are expected to be the result of adaptive evolution, the features of these networks may provide the prokaryotic taxonomist with novel tools for reaching the fourth tenet of microbial systematics. Through selected examples from the Actinobacteria, which have been inferred from GSMRs and experimentally confirmed after phenotypic characterisation, it will be shown that this level of information can be incorporated into modern polyphasic taxonomic approaches. In conclusion, three specific examples are illustrated to show how GSMRs will revolutionize prokaryotic systematics, as has previously occurred in many other fields of microbiology.     

Chemodiversity Associated with Cytotoxicity and Antimicrobial Activity of Piper aduncum var. ossanum

Chemical analysis, antimicrobial activity and cytotoxic effects of essential oils (EOs) from leaves of Piper aduncum var. ossanum from two localities Bauta (EO‐B) and Ceiba (EO‐C), Artemisa Province, Cuba, were determined. EOs were obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry. EO‐B demonstrated higher activity against S. aureus and L. amazonensis; while a lower cytotoxicity on mammalian cells was observed. Both EOs displayed the same activity against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei, and Leishmania infantum. Both EOs were inactive against Escherichia coli and Candida albicans.     

Identification and analysis of residues contained on β → α loops of the dual‐substrate (βα)8 phosphoribosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity

A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient‐like dual‐substrate (βα)₈ phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis–Menten enzyme kinetics for both isomerase activities in wild‐type PriA from S. coelicolor and in selected single‐residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg¹³⁹, which was postulated on structural grounds to be important for the dual‐substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser⁸¹Thr and PriA_Arg¹³⁹Asn showed that these residues, which are contained on β → α loops and in close proximity to the N‐terminal phosphate‐binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X‐ray crystallographic structure of PriA_Arg¹³⁹Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α loop as a major structural feature related to the evolution of the dual‐substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates—within a bifunctional and thus highly constrained active site—without compromising its structural stability.     

The structure/function relationship of a dual-substrate (betaalpha)8-isomerase

Two structures of phosphoribosyl isomerase A (PriA) from Streptomyces coelicolor, involved in both histidine and tryptophan biosynthesis, were solved at 1.8 Å resolution. A closed conformer was obtained, which represents the first complete structure of PriA, revealing hitherto unnoticed molecular interactions and the occurrence of conformational changes. Inspection of these conformers, including ligand-docking simulations, allowed identification of residues involved in substrate recognition, chemical catalysis and conformational changes. These predictions were validated by mutagenesis and functional analysis. Arg¹⁹ and Ser⁸¹ were shown to play critical roles within the carboxyl and amino phosphate-binding sites, respectively; the catalytic residues Asp¹¹ and Asp¹³⁰ are responsible for both activities; and Thr¹⁶⁶ and Asp¹⁷¹, which make an unusual contact, are likely to elicit the conformational changes needed for adopting the active site architectures. This represents the first report of the structure/function relationship of this (βα)₈-isomerase.     

The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC₅₀) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC₅₀ and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural^(r) (a furvina-containing antifungal ointment) for the treatment of CL.     

Antibiotic resistance evolved via inactivation of a ribosomal RNA methylating enzyme

Modifications of the bacterial ribosome regulate the function of the ribosome and modulate its susceptibility to antibiotics. By modifying a highly conserved adenosine A2503 in 23S rRNA, methylating enzyme Cfr confers resistance to a range of ribosome-targeting antibiotics. The same adenosine is also methylated by RlmN, an enzyme widely distributed among bacteria. While RlmN modifies C2, Cfr modifies the C8 position of A2503. Shared nucleotide substrate and phylogenetic relationship between RlmN and Cfr prompted us to investigate evolutionary origin of antibiotic resistance in this enzyme family. Using directed evolution of RlmN under antibiotic selection, we obtained RlmN variants that mediate low-level resistance. Surprisingly, these variants confer resistance not through the Cfr-like C8 methylation, but via inhibition of the endogenous RlmN C2 methylation of A2503. Detection of RlmN inactivating mutations in clinical resistance isolates suggests that the mechanism used by the in vitro evolved variants is also relevant in a clinical setting. Additionally, as indicated by a phylogenetic analysis, it appears that Cfr did not diverge from the RlmN family but from another distinct family of predicted radical SAM methylating enzymes whose function remains unknown.     

SH3 interactome conserves general function over specific form

Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two‐hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain‐mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.