Total chemical synthesis of lassomycin and lassomycin-amide.

Herein we report a practical synthetic route to the lasso peptide lassomycin () and C-terminal variant lassomycin-amide (). The biological evaluation of peptides and against Mycobacterium tuberculosis revealed that neither had any activity against this bacterium. This lack of biological activity has led us to propose that naturally occurring lassomycin may actually exhibit a standard lasso peptide threaded conformation rather than the previously reported unthreaded structure.

Pestalone, a new antibiotic produced by a marine fungus in response to bacterial challenge.

The isolation and structure determination of a new chlorinated benzophenone antibiotic, pestalone (1), is described. The new compound was produced by a cultured marine fungus only when a unicellular marine bacterium, strain CNJ-328, was co-cultured in the fungal fermentation. The fungus, isolated from the surface of the brown alga Rosenvingea sp. collected in the Bahamas Islands, was identified as an undescribed member of the genus Pestalotia. The structure of 1, initially assigned with only modest confidence by combined spectral and chemical data, was confirmed by single-crystal X-ray analysis. Pestalone (1) exhibits moderate in vitro cytotoxicity in the National Cancer Institute's 60 human tumor cell line screen (mean GI(50) = 6.0 microM). More importantly, pestalone shows potent antibiotic activity against methicillin-resistant Staphylococcus aureus (MIC = 37 ng/mL) and vancomycin-resistant Enterococcus faecium (MIC = 78 ng/mL), indicating that pestalone should be evaluated in advanced models of infectious disease.

Synthesis and biological evaluation of analogues of the antibiotic pantocin B.

Strains of the bacteria Erwinia herbicola produce antibiotics that effectively control E. amylovora, the bacterial pathogen responsible for the plant disease fire blight. Pantocin B was the first of these antibiotics to be characterized, and a flexible synthesis of various analogues is reported. Embedded in the "pseudo-tripeptide" backbone of pantocin B are a methylenediamine and a methyl sulfone, both unusual structural features in natural products. The peptidic nature of pantocin B facilitated a series of structure-activity relationship studies that probed the roles of these functional groups in determining the biological activity of pantocin B. A clear demarcation of the roles between the N- and C-terminal portions of the antibiotic was determined as a result of the structure-activity relationship studies. The N-terminal L-alanyl group is needed for cellular import but not for interaction with the intracellular target, the arginine biosynthetic enzyme N-acetylornithine aminotransferase. The methylenediamine and methyl sulfone portions were found to be essential for antibiotic activity, presumably due to extensive interactions with N-acetylornithine aminotransferase.

Dicerandrols, new antibiotic and cytotoxic dimers produced by the fungus Phomopsis longicolla isolated from an endangered mint.

Bioassay-guided fractionation of the organic extract from a culture of Phomopsis longicolla, an endophytic fungus of the endangered mint Dicerandra frutescens, led to the isolation of dicerandrols A, B, and C. Extensive NMR and HRFABMS experiments were used to identify these new yellow antibiotic and cytotoxic compounds as 2,2'-dimeric tetrahydroxanthones.

Cloning and heterologous expression of a natural product biosynthetic gene cluster from eDNA.

[reaction: see text] To study the natural products produced by uncultured microorganisms, an environmental DNA (eDNA) cosmid library was constructed and screened for the heterologous production of small molecules. A blue clone, CSL51, found in the eDNA library produces deoxyviolacein and the broad spectrum antibiotic violacein. The full sequence of the 6.7 kb eDNA violacein gene cluster and the characterization of violacein and deoxyviolacein from an eDNA clone are reported here.

A pregnane steroid from Aglaia lawii and structure confirmation of cabraleadiol monoacetate by X-ray crystallography.

The pregnane steroid, (E)-aglawone, along with four known triterpenes, and a known sterol mixture were isolated from the bark of Aglaia lawii (Wight) Saldanha ex Ramamoorty (Meliaceae). The structural determination/identification was accomplished by a combination of 1D- and 2D-NMR spectroscopic techniques. The relative stereochemistry of the known triterpene, 20S,24S-epoxydammarane-3alpha,25-diol acetate, was also unequivocally determined for the first time by X-ray crystallography. The isolates were not active against various human cancer cell lines.

The cytosporones, new octaketide antibiotics isolated from an endophytic fungus.

[structure] Organic extracts from cultures of endophytic fungi collected in the Guanacaste Conservation Area of Costa Rica were screened for antibiotic activity. Two endophytes CR200 (Cytospora sp.) and CR146 (Diaporthe sp.) were found to have potent antibiotic activity. Bioassay-guided fractionation of the extracts from these fungi led to the identification of cytosporones D and E, antibacterial active trihydroxybenzene lactones, and three related but inactive metabolites. The five new octaketides were characterized using X-ray crystallography and NMR.

Cytoskyrins A and B, new BIA active bisanthraquinones isolated from an endophytic fungus.

[structure] The biochemical induction assay (BIA) is a rapid (colorimetric) bacterial assay used to identify compounds that damage DNA or inhibit DNA synthesis and thereby identify potential natural product anticancer agents. Bisanthraquinones based on a 1,3,6, 8-tetrahydroxyanthraquinone-type carbon skeleton were isolated from an endophytic fungus and characterized by NMR and X-ray crystallography. Cytoskyrin A (1) is highly active in the biochemical induction assay, while the closely related cytoskyrin B (2) has no detectable activity in this assay.

Differences in medical students' empathy.

Medical students' Balanced Emotional Empathy Scale scores were compared by year, sex, and expressed specialty choice. Scores were lower for students choosing non-core specialties, and for M4 men students compared with M3 men students.

CR377, a new pentaketide antifungal agent isolated from an endophytic fungus.

Cultures of endophytic fungi collected in the Guanacaste Conservation Area of Costa Rica were screened for antifungal activity. CR377, a new pentaketide antifungal agent, was isolated from the culture broth of a fungus, CR377 (Fusarium sp.), that showed potent activity against Candida albicans in this assay. The structure of CR377 was established using 1- and 2-D NMR and HRFABMS.

Honulactones: new bishomoscalarane sesterterpenes from the Indonesian sponge Strepsichordaia aliena.

From the dichloromethane/2-propanol (1:1) extract of the Indonesian marine sponge Strepsichordaia aliena, twelve new 20, 24-bishomoscalarane sesterterpenes, honulactones A-L (1-12) were isolated. Molecular structures were secured by spectroscopic methods, accurate mass measurements, and X-ray analysis. Honulactones A (1), B (2), C (3), and D (4) exhibit cytotoxycity against P-388, A-549, HT-29, and MEL-28 (IC(50) 1 microg/mL).

Rhizobium etli CE3 carries vir gene homologs on a self-transmissible plasmid.

RosR is a transcriptional regulator important for determining cell-surface characteristics and nodulation competitiveness in Rhizobium etli CE3. We identified a 15-kb region that contains genes with similarity to members of the virB, virC, virG, and virE operons of Agrobacterium tumefaciens and demonstrated that RosR directly regulates one operon in this region. These genes were located on plasmid pa of R. etli CE3, which is self-transmissible between R. etli and A. tumefaciens.

Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride.

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.

The total synthesis of pantocin B.

[reaction: see text] Pantocin B, an unusual antibiotic produced by Erwinia herbicola, effectively controls E. amylovora, the pathogen causing the plant disease fire blight. A total synthesis of pantocin B from L-alanine, glycine, and L-malic acid is reported.

Biological activity of guanacastepene, a novel diterpenoid antibiotic produced by an unidentified fungus CR115.

Fermentation extracts of culture CR115, an unknown plant endophyte originally isolated from Costa Rica, were found to be active against antibiotic-resistant bacteria. The metabolite responsible for activity was identified as a novel diterpenoid antibiotic guanacastepene (mol. wt. 374.47 and mol. formula C22H30O5). Mechanistic studies done in an E. coli imp strain suggested membrane damage as the primary mode of bactericidal action. This compound also lysed human RBCs and caused leakage of intracellular potassium from E. coli imp.

The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis.

Mutations in palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme that removes fatty acyl groups from cysteine residues in modified proteins, cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis. The accumulation of undigested substrates leads to the formation of neuronal storage bodies that are associated with the clinical symptoms. Less severe forms of PPT1 deficiency have been found recently that are caused by a distinct set of PPT1 mutations, some of which retain a small amount of thioesterase activity. We have determined the crystal structure of PPT1 with and without bound palmitate by using multiwavelength anomalous diffraction phasing. The structure reveals an alpha/beta-hydrolase fold with a catalytic triad composed of Ser115-His289-Asp233 and provides insights into the structural basis for the phenotypes associated with PPT1 mutations.

Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein-bombykol complex.

BACKGROUND: Insects use volatile organic molecules to communicate messages with remarkable sensitivity and specificity. In one of the most studied systems, female silkworm moths (Bombyx mori) attract male mates with the pheromone bombykol, a volatile 16-carbon alcohol. In the male moth's antennae, a pheromone-binding protein conveys bombykol to a membrane-bound receptor on a nerve cell. The structure of the pheromone-binding protein, its binding and recognition of bombykol, and its full role in signal transduction are not known. RESULTS: The three-dimensional structure of the B. mori pheromone-binding protein with bound bombykol has been determined by X-ray diffraction at 1.8 A resolution. CONCLUSIONS: The pheromone binding protein of B. mori has six helices, and bombykol binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. Bombykol is bound in this cavity through numerous hydrophobic interactions, and sequence alignments suggest critical residues for specific pheromone binding.