19 research outputs found
Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in Streptomyces coelicolor A3(2)*Sâ
Cytochrome P450 170A1 (CYP170A1) is encoded by the sco5223 gene of
the Gram-positive, soil-dwelling bacterium Streptomyces coelicolor
A3(2) as part of a two-gene cluster with the sco5222 gene. The
SCO5222 protein is a sesquiterpene synthase that catalyzes the cyclization of
farnesyl diphosphate to the novel tricyclic hydrocarbon, epi-isozizaene (Lin,
X., Hopson, R., and Cane, D. E. (2006) J. Am. Chem.
Soc. 128, 6022â6023). The presence of CYP170A1
(sco5223) suggested that epiisozizaene might be further oxidized by
the transcriptionally coupled P450. We have now established that purified
CYP170A1 carries out two sequential allylic oxidations to convert
epi-isozizaene to an epimeric mixture of albaflavenols and thence to the
sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry
analysis of S. coelicolor culture extracts established the presence
of albaflavenone in the wild-type strain, along with its precursors
epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene
abolished biosynthesis of both albaflavenone and the albaflavenols, but not
epi-isozizaene. The combined results establish for the first time the presence
of albaflavenone in S. coelicolor and clearly demonstrate that the
biosynthesis of this antibiotic involves the coupled action of epi-isozizaene
synthase and CYP170A1
Solving the alpha-conotoxin folding problem: Efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antaqonists
alpha-Conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 angstrom resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha 3 beta 4, alpha 7, and alpha 1 beta 1 delta gamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins
Resin-bound sulfonyl azides: efficient loading and activation strategy for the preparation of theN-Acyl sulfonamide linker
This paper describes an optimized protocol for the efficient loading of resin-bound aminoethane sulfonyl azides by either Boc- or Fmoc-protected amino thioacids. The resulting N-acyl sulfonamide is a convenient linker for use in Boc- or Fmoc-based solid-phase peptide synthesis. Activation of the N-acyl sulfonamide via a microwave-assisted alkylation procedure and subsequent treatment with functionalized nucleophiles yields C-terminally modified peptides that can be applied in chemoselective (bio)conjugation or ligation reactions