19 research outputs found
Chemical Perturbation of Secondary Metabolism Demonstrates Important Links to Primary Metabolism
SummaryBacterially produced secondary metabolites are used as antibiotics, anticancer drugs, and for many other medicinal applications. The mechanisms that limit the production of these molecules in the laboratory are not well understood, and this has impeded the discovery of many important compounds. We have identified small molecules that remodel the yields of secondary metabolites in many actinomycetes and show that one set of these molecules does so by inhibiting fatty acid biosynthesis. This demonstrates a particularly intimate relationship between this primary metabolic pathway and secondary metabolism and suggests an approach to enhance the yields of metabolites for discovery and biochemical characterization
Carbon Monoxide-Releasing Molecule-401 Suppresses Polymorphonuclear Leukocyte Migratory Potential by Modulating F-Actin Dynamics
Carbon monoxide-releasing molecules (CORMs) suppress inflammation by reducing polymorphonuclear leukocyte (PMN) recruitment to the affected organs. We investigated modulation of PMN-endothelial cell adhesive interactions by water-soluble CORM-401 using an experimental model of endotoxemia in vitro. Human umbilical vein endothelial cells (HUVEC) grown on laminar-flow perfusion channels were stimulated with 1 μg/mL lipopolysaccharide for 6 hours and perfused with 100 μmol/L CORM-401 (or inactive compound iCORM-401)-pretreated PMN for 5 minutes in the presence of 1.0 dyn/cm
HUVEC: PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMN with CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P \u3c 0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distribution and fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P \u3c 0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophil migratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics
A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer
Abstract Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 – 100 nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases
Transition State Analysis of Enolpyruvylshikimate 3‑Phosphate (EPSP) Synthase (AroA)-Catalyzed EPSP Hydrolysis
Proton transfer to carbon atoms is a significant catalytic
challenge
because of the large intrinsic energetic barrier and the frequently
unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate
3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene
carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction.
We performed transition state analysis using kinetic isotope effects
(KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a
methylene carbon (C3) protonation, as an analog of AroA’s reverse
reaction. As part of this analysis, an inorganic phosphate scavenging
system was developed to remove phosphate which, though present in
microscopic amounts in solution, is ubiquitous. The reaction was stepwise,
with irreversible C3 protonation to form an EPSP cation intermediate;
that is, an A<sub>H</sub><sup>‡</sup>*A<sub>N</sub> mechanism.
The large experimental 3-<sup>14</sup>C KIE, 1.032 ± 0.005, indicated
strong coupling of C3 with the motion of the transferring proton.
Calculated 3-<sup>14</sup>C KIEs for computational transition state
models revealed that the transition state occurs early during C3–H<sup>+</sup> bond formation, with a C3–H<sup>+</sup> bond order
of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04,
was the lowest observed to date for this type of reaction, but consistent
with a very early transition state. The large 2-<sup>14</sup>C KIE
reflected an “electrostatic sandwich” formed by Asp313
and Glu341 to stabilize the positive charge at C2. In shifting the
transition state earlier than the acid-catalyzed reaction, AroA effected
a large Hammond shift, indicating that a significant part of AroA’s
catalytic strategy is to stabilize the positive charge in the EPSP
cation. A computational model containing all the charged amino acid
residues in the AroA active site close to the reactive center showed
a similar Hammond shift relative to the small transition state models
Transition State Analysis of Acid-Catalyzed Hydrolysis of an Enol Ether, Enolpyruvylshikimate 3‑Phosphate (EPSP)
Proton transfer to carbon represents a significant catalytic
challenge
because of the large intrinsic energetic barrier and the frequently
unfavorable thermodynamics. Multiple kinetic isotope effects (KIEs)
were measured for acid-catalyzed hydrolysis of the enol ether functionality
of enolpyruvylshikimate 3-phosphate (EPSP) as a nonenzymatic analog
of the EPSP synthase (AroA) reaction. The large solvent deuterium
KIE demonstrated that protonating C3 was the rate-limiting step, and
the lack of solvent hydron exchange into EPSP demonstrated that protonation
was irreversible. The reaction mechanism was stepwise, with C3, the
methylene carbon, being protonated to form a discrete oxacarbenium
ion intermediate before water attack at the cationic center, that
is, an A<sub>H</sub><sup>‡</sup>*A<sub>N</sub> (or A<sub>H</sub><sup>‡</sup> + A<sub>N</sub>) mechanism. The calculated 3-<sup>14</sup>C and 3,3-<sup>2</sup>H<sub>2</sub> KIEs varied as a function
of the extent of proton transfer at the transition state, as reflected
in the C3–H<sup>+</sup> bond order, <i>n</i><sub>C3–H+</sub>. The calculated 3-<sup>14</sup>C KIE was a function
primarily of C3 coupling with the movement of the transferring proton,
as reflected in the reaction coordinate contribution (<sup>light</sup>ν<sup>‡</sup>/<sup>heavy</sup>ν<sup>‡</sup>), rather than of changes in bonding. Coupling was strongest in early
and late transition states, where the reaction coordinate frequency
was lower. The other calculated <sup>14</sup>C and <sup>18</sup>O
KIEs were more sensitive to interactions with counterions and solvation
in the model structures than <i>n</i><sub>C3–H+</sub>. The KIEs revealed a moderately late transition state with significant
oxacarbenium ion character and with a C3–H<sup>+</sup> bond
order ≈0.6
Metabolomic-guided discovery of cyclic nonribosomal peptides from Xylaria ellisii sp. nov., a leaf and stem endophyte of Vaccinium angustifolium
Fungal endophytes are sources of novel bioactive compounds but relatively few agriculturally important fruiting plants harboring endophytes have been carefully studied. Previously, we identified a griseofulvin-producing Xylaria species isolated from Vaccinium angustifolium, V. corymbosum, and Pinus strobus. Morphological and genomic analysis determined that it was a new species, described here as Xylaria ellisii. Untargeted high-resolution LC-MS metabolomic analysis of the extracted filtrates and mycelium from 15 blueberry isolates of this endophyte revealed differences in their metabolite profiles. Toxicity screening of the extracts showed that bioactivity was not linked to production of griseofulvin, indicating this species was making additional bioactive compounds. Multivariate statistical analysis of LC-MS data was used to identify key outlier features in the spectra. This allowed potentially new compounds to be targeted for isolation and characterization. This approach resulted in the discovery of eight new proline-containing cyclic nonribosomal peptides, which we have giv