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1.
Sci Rep ; 13(1): 11944, 2023 07 24.
Article in English | MEDLINE | ID: mdl-37488207

ABSTRACT

Chrysophaeum taylorii is a member of an understudied clade of marine algae that can be responsible for harmful coastal blooms and is known to accumulate bioactive natural products including antibiotics of the chrysophaentin class. Whole genome sequencing of laboratory-cultivated samples revealed an extensive and diverse complement of secondary metabolite biosynthetic genes in C. taylorii, alongside a small microbiome with a more limited biosynthetic potential. 16S microbiome analysis of laboratory cultured alongside wild-collected samples revealed several common taxa; however, analysis of biosynthetic genes suggested an algal origin for the chrysophaentins, possibly via one of several non-canonical polyketide synthase genes encoded within the genome.


Subject(s)
Biological Products , Microbiota , Secondary Metabolism , Anti-Bacterial Agents , Laboratories
2.
Molecules ; 26(23)2021 Nov 27.
Article in English | MEDLINE | ID: mdl-34885778

ABSTRACT

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.


Subject(s)
Actinomycetales/chemistry , Pyrazines/isolation & purification , Soil Microbiology , Biosynthetic Pathways/genetics , Carbon-13 Magnetic Resonance Spectroscopy , Genome, Bacterial , Multigene Family , Peptide Synthases/metabolism , Proton Magnetic Resonance Spectroscopy , Substrate Specificity
3.
Org Lett ; 23(3): 682-686, 2021 02 05.
Article in English | MEDLINE | ID: mdl-33444500

ABSTRACT

Six novel pyranonaphthoquinones, vertirhodins A-F (1-6), were discovered from a soil-derived Streptomyces sp. B15-008. Their chemical structures and absolute configurations were determined using nuclear magnetic resonance and comparison of experimental and theoretical electronic circular dichroism spectra. The vertirhodins feature an unusual C-8 N-methyl-2-pyrrolidinemethanol moiety, a 5,14-epoxide rarely seen in streptomyces-derived natural products, and a C-13 hydroxyl group that forms the semiquinone. A plausible ver biosynthetic gene cluster was identified through whole genome sequencing and provides insights into these features.


Subject(s)
Biological Products/chemistry , Naphthoquinones/chemistry , Pyrrolidines/chemistry , Streptomyces/chemistry , Circular Dichroism , Magnetic Resonance Spectroscopy , Molecular Structure , Multigene Family , Naphthoquinones/isolation & purification , Pyrrolidines/isolation & purification
4.
J Nat Prod ; 82(1): 148-153, 2019 01 25.
Article in English | MEDLINE | ID: mdl-30623657

ABSTRACT

A laboratory culture of the colonial marine alga Chrysophaeum taylorii NIES-1699 yielded a set of new bioactive chrysophaentin analogs, and their structures were determined by HRESIMS and NMR spectroscopy. Differences in the metabolites identified between cultured C. taylorii NIES-1699 and field-collected strains from the U.S. Virgin Islands revealed additional structure-activity relationships for the Gram-positive antibiotic activity of the chrysophaentins. The presence of new hemichrysophaentins and a C-C linked biphenyl analog suggest novel features of their biosynthetic pathway. Bayesian analysis of the alignment of the 18S rRNA gene places the microalga C. taylorii in the pelagophyte clade.


Subject(s)
Anti-Bacterial Agents/isolation & purification , Biphenyl Compounds/isolation & purification , Microalgae/metabolism , Anti-Bacterial Agents/pharmacology , Bayes Theorem , Benzyl Compounds , Biphenyl Compounds/metabolism , Biphenyl Compounds/pharmacology , Ethers, Cyclic , Marine Biology , Structure-Activity Relationship
5.
Methods Enzymol ; 598: 3-35, 2018.
Article in English | MEDLINE | ID: mdl-29306440

ABSTRACT

Lectins are carbohydrate-binding proteins unrelated to antibodies or enzymes. While carbohydrates are present on all cells and pathogens, lectins are also ubiquitous in nature and their interactions with glycans mediate countless biological and physical interactions. Due to the multivalency found in both lectins and their glycan-binding partners, complete characterization of these interactions can be complex and typically requires the use of multiple complimentary techniques. In this chapter, we provide a general strategy and protocols for chemical and biophysical approaches that can be used to characterize carbohydrate-mediated interactions in the context of individual oligosaccharides, as part of a glycoprotein, and ending with visualization of interactions with whole virions.


Subject(s)
Glycoproteins/chemistry , Lectins/chemistry , Oligosaccharides/chemistry , Virion/metabolism , Calorimetry/instrumentation , Calorimetry/methods , Dynamic Light Scattering/instrumentation , Dynamic Light Scattering/methods , Glycopeptides/chemical synthesis , Glycopeptides/metabolism , Glycoproteins/metabolism , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , Lectins/genetics , Lectins/isolation & purification , Lectins/metabolism , Nuclear Magnetic Resonance, Biomolecular/instrumentation , Nuclear Magnetic Resonance, Biomolecular/methods , Oligosaccharides/metabolism , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Surface Plasmon Resonance/instrumentation , Surface Plasmon Resonance/methods , Ultracentrifugation/instrumentation , Ultracentrifugation/methods
6.
Nat Chem Biol ; 14(2): 108-109, 2018 01 16.
Article in English | MEDLINE | ID: mdl-29337972
7.
Article in English | MEDLINE | ID: mdl-28373194

ABSTRACT

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Biological Transport/genetics , Biological Transport/physiology , Genes, MDR/genetics , Genes, MDR/physiology , Microbial Sensitivity Tests , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Nucleosides/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity
8.
Chembiochem ; 18(8): 764-771, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28166380

ABSTRACT

Man9 GlcNAc2 (Man-9) present at the surface of HIV makes up the binding sites of several HIV-neutralizing agents and the mammalian lectin DC-SIGN, which is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in its free state and when bound by the HIV entry-inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC-SIGN by using NMR spectroscopy. To facilitate the implementation of 3D 13 C-edited spectra to deconvolute spectral overlap and to determine the solution structure of Man-9, we developed a robust expression system for the production of 13 C,15 N-labeled glycans in mammalian cells. The studies reveal that Man-9 interacts with HIV-binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor-bound conformations to be sampled by Man-9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.


Subject(s)
Bacterial Proteins/chemistry , Cell Adhesion Molecules/chemistry , Lectins, C-Type/chemistry , Magnetic Resonance Spectroscopy , Mannans/chemistry , Mannose-Binding Lectin/chemistry , Receptors, Cell Surface/chemistry , A549 Cells , Carbohydrate Conformation , Carbon Radioisotopes , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Humans , Mannans/biosynthesis , Microcystis , Molecular Dynamics Simulation , Nitrogen Radioisotopes
9.
Cell ; 165(4): 813-26, 2016 May 05.
Article in English | MEDLINE | ID: mdl-27114034

ABSTRACT

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.


Subject(s)
HIV-1/chemistry , env Gene Products, Human Immunodeficiency Virus/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Crystallography, X-Ray , Glycosylation , HIV-1/classification , HIV-1/immunology , Immune Evasion , Models, Molecular , Molecular Dynamics Simulation , Polysaccharides/analysis , Polysaccharides/metabolism
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