13 research outputs found
Generation of Novel Pikromycin Antibiotic Products Through Mutasynthesis
Mutasynthesis in pikromycin PKS: The amenability of pikromycin polyketide synthase to mutational biosynthesis has been demonstrated. A natural triketide and its analogues, activated as N-acetyl-cysteamine thioesters, were synthesized and fed to a pikAI-deleted strain; this led to the production of new antibiotics. A vinyl analogue was found to have better antibacterial activity than pikromycin
Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase
Metabolic
engineering of polyketide synthase (PKS) pathways represents a promising
approach to natural products discovery. The dehydratase (DH) domains
of PKSs, which generate an α,β-unsaturated bond through
a dehydration reaction, have been poorly studied compared with other
domains, likely because of the simple nature of the chemical reaction
they catalyze and the lack of a convenient assay to measure substrate
turnover. Herein we report the first steady-state kinetic analysis
of a PKS DH domain employing LC–MS/MS analysis for product
quantitation. PikDH2 was selected as a model DH domain. Its substrate
specificity and mechanism were interrogated with a systematic series
of synthetic triketide substrates containing a nonhydrolyzable thioether
linkage as well as by site-directed mutagenesis, evaluation of the
pH dependence of the catalytic efficiency (<i>V</i><sub>max</sub>/<i>K</i><sub>M</sub>), and kinetic characterization
of a mechanism-based inhibitor. These studies revealed that PikDH2
converts d-alcohol substrates to <i>trans</i>-olefin
products. The reaction is reversible with equilibrium constants ranging
from 1.2 to 2. Moreover, the enzyme activity is robust, and PikDH2
was used on a preparative scale for the chemoenzymatic synthesis of
unsaturated triketide products. PikDH2 was shown to possess remarkably
strict substrate specificity and is unable to turn over substrates
that are epimeric at the β-, γ-, or δ-position.
We also demonstrated that PikDH2 has a key ionizable group with a
p<i>K</i><sub>a</sub> of 7.0 and can be irreversibly inactivated
through covalent modification by a mechanism-based inhibitor, which
provides a foundation for future structural studies to elucidate substrate–protein
interactions
Polyketide Intermediate Mimics as Probes for Revealing Cryptic Stereochemistry of Ketoreductase Domains
Among
natural product families, polyketides have shown the most
promise for combinatorial biosynthesis of natural product-like libraries.
Though recent research in the area has provided many mechanistic revelations,
a basic-level understanding of kinetic and substrate tolerability
is still needed before the full potential of combinatorial biosynthesis
can be realized. We have developed a novel set of chemical probes
for the study of ketoreductase domains of polyketide synthases. This
chemical tool-based approach was validated using the ketoreductase
of pikromycin module 2 (PikKR2) as a model system. Triketide substrate
mimics <b>12</b> and <b>13</b> were designed to increase
stability (incorporating a nonhydrolyzable thioether linkage) and
minimize nonessential functionality (truncating the phosphopantetheinyl
arm). PikKR2 reduction product identities as well as steady-state
kinetic parameters were determined by a combination of LC-MS/MS analysis
of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results
from a complementary biochemical and molecular biological approach.
When compared to widely employed substrates in previous studies, diketide <b>63</b> and <i>trans</i>-decalone <b>64</b>, substrates <b>12</b> and <b>13</b> showed 2–10 fold lower <i>K</i><sub>M</sub> values (2.4 ± 0.8 and 7.8 ± 2.7
mM, respectively), indicating molecular recognition of intermediate-like
substrates. Due to an abundance of the nonreducable enol-tautomer,
the <i>k</i><sub>cat</sub> values were attenuated by as
much as 15–336 fold relative to known substrates. This study
reveals the high stereoselectivity of PikKR2 in the face of gross
substrate permutation, highlighting the utility of a chemical probe-based
approach in the study of polyketide ketoreductases
Discovery of a potent, selective, and efficacious class of reversible alpha-ketoheterocycle inhibitors of fatty acid amide hydrolase effective as analgesics
Fatty acid amide hydrolase (FAAH) degrades neuromodulating fatty acid amides including anandamide (endogenous cannabinoid agonist) and oleamide (sleep-inducing lipid) at their sites of action and is intimately involved in their regulation. Herein we report the discovery of a potent, selective, and efficacious class of reversible FAAH inhibitors that produce analgesia in animal models validating a new therapeutic target for pain intervention. Key to the useful inhibitor discovery was the routine implementation of a proteomics-wide selectivity screen against the serine hydrolase superfamily ensuring selectivity for FAAH coupled with systematic in vivo examinations of candidate inhibitors