9 research outputs found
Combinatorialization of Fungal Polyketide Synthase–Peptide Synthetase Hybrid Proteins
The
programming of the fungal polyketide synthase (PKS) is quite
complex, with a simple domain architecture leading to elaborate products.
An additional level of complexity has been found within PKS-based
pathways where the PKS is fused to a single module nonribosomal peptide
synthetase (NRPS) to synthesize polyketides conjugated to amino acids.
Here, we sought to understand the communication between these modules
that enable correct formation of polyketide-peptide hybrid products.
To do so, we fused together the genes that are responsible for forming
five highly chemically diverse fungal natural products in a total
of 57 different combinations, comprising 34 distinct module swaps.
Gene fusions were formed with the idea of testing the connection and
compatibility of the PKS and NRPS modules mediated by the acyl carrier
protein (ACP), condensation (C) and ketoreductase (KR) domains. The
resulting recombinant gene fusions were analyzed in a high-yielding
expression platform to avail six new compounds, including the first
successful fusion between a PKS and NRPS that make highly divergent
products, and four previously reported molecules. Our results show
that C domains are highly selective for a subset of substrates. We
discovered that within the highly reducing (hr) PKS class, noncognate
ACPs of closely related members complement PKS function. We intercepted
a pre-Diels–Alder intermediate in lovastatin synthesis for
the first time, shedding light on this canonical fungal biochemical
reaction. The results of these experiments provide a set of ground
rules for the successful engineering of hr-PKS and PKS-NRPS products
in fungi
Two Related Pyrrolidinedione Synthetase Loci in <i>Fusarium heterosporum</i> ATCC 74349 Produce Divergent Metabolites
Equisetin
synthetase (EqiS), from the filamentous fungus <i>Fusarium heterosporum</i> ATCC 74349, was initially assigned
on the basis of genetic knockout and expression analysis. Increasing
inconsistencies in experimental results led us to question this assignment.
Here, we sequenced the <i>F. heterosporum</i> genome, revealing
two hybrid polyketide-peptide proteins that were candidates for the
equisetin synthetase. The surrounding genes in both clusters had the
needed auxiliary genes that might be responsible for producing equisetin.
Genetic mutation, biochemical analysis, and recombinant expression
in the fungus enabled us to show that the initially assigned EqiS
does not produce equisetin but instead produces a related 2,4-pyrrolidinedione,
fusaridione A, that was previously unknown. Fusaridione A is methylated
in the 3-position of the pyrrolidinedione, which has not otherwise
been found in natural products, leading to spontaneous reverse-Dieckmann
reactions. A newly described gene cluster, <i>eqx</i>, is
responsible for producing equisetin
Aestuaramides, a Natural Library of Cyanobactin Cyclic Peptides Resulting from Isoprene-Derived Claisen Rearrangements
We
report 12 cyanobactin cyclic peptides, the aestuaramides, from
the cultivated cyanobacterium <i>Lyngbya aestuarii</i>.
We show that aestuaramides are synthesized enzymatically as reverse <i>O</i>-prenylated tyrosine ethers that subsequently undergo a
Claisen rearrangement to produce forward <i>C</i>-prenylated
tyrosine. These results reveal that a nonenzymatic Claisen rearrangement
dictates isoprene regiochemistry in a natural system. They also reveal
one of the mechanisms that organisms use to generate structurally
diverse compound libraries starting from simple ribosomal peptide
pathways (RiPPs)
Enzymatic N- and C‑Protection in Cyanobactin RiPP Natural Products
Recent innovations in peptide natural
product biosynthesis reveal
a surprising wealth of previously uncharacterized biochemical reactions
that have potential applications in synthetic biology. Among these,
the cyanobactins are noteworthy because these peptides are protected
at their N- and C-termini by macrocyclization. Here, we use a novel
bifunctional enzyme AgeMTPT to protect linear peptides by attaching
prenyl and methyl groups at their free N- and C-termini. Using this
peptide protectase in combination with other modular biosynthetic
enzymes, we describe the total synthesis of the natural product aeruginosamide
B and the biosynthesis of linear cyanobactin natural products. Our
studies help to define the enzymatic mechanism of macrocyclization,
providing evidence against the water exclusion hypothesis of transpeptidation
and favoring the kinetic lability hypothesis
Small Molecules in the Cone Snail Arsenal
Cone
snails are renowned for producing peptide-based venom, containing
conopeptides and conotoxins, to capture their prey. A novel small-molecule
guanine derivative with unprecedented features, genuanine, was isolated
from the venom of two cone snail species. Genuanine causes paralysis
in mice, indicating that small molecules and not just polypeptides
may contribute to the activity of cone snail venom
Modulating the Serotonin Receptor Spectrum of Pulicatin Natural Products
Serotonin (5-HT) receptors are important
in health and disease,
but the existence of 14 subtypes necessitates selective ligands. Previously,
the pulicatins were identified as ligands that specifically bound
to the subtype 5-HT<sub>2B</sub> in the 500 nM to 10 μM range and that exhibited <i>in
vitro</i> effects on cultured mouse neurons. Here, we examined
the structure–activity relationship of 30 synthetic and natural
pulicatin derivatives using binding, receptor functionality, and <i>in vivo</i> assays. The results reveal the 2-arylthiazoline
scaffold as a tunable serotonin receptor-targeting pharmacophore.
Tests in mice show potential antiseizure and antinociceptive activities
at high doses without motor impairment
Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis
Prenylation is a widespread modification
that improves the biological
activities of secondary metabolites. This reaction also represents
a key modification step in biosyntheses of cyanobactins, a family
of ribosomally synthesized and post-translationally modified peptides
(RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are
commonly isoprenylated by ABBA prenyltransferases that use C<sub>5</sub> donors. Notably, mass spectral analysis of piricyclamides from a
fresh-water cyanobacterium suggested that they may instead have a
C<sub>10</sub> geranyl group. Here we characterize a novel geranyltransferase
involved in piricyclamide biosynthesis. Using the purified enzyme,
we show that the enzyme PirF catalyzes Tyr <i>O</i>-geranylation,
which is an unprecedented post-translational modification. In addition,
the combination of enzymology and analytical chemistry revealed the
structure of the final natural product, piricyclamide 7005E1, and
the regioselectivity of PirF, which has potential as a synthetic biological
tool providing drug-like properties to diverse small molecules
Oxazinin A, a Pseudodimeric Natural Product of Mixed Biosynthetic Origin from a Filamentous Fungus
A racemic,
prenylated polyketide dimer, oxazinin A (<b>1</b>), was isolated
from a novel filamentous fungus in the class Eurotiomycetes,
and its structure was elucidated spectroscopically. The pentacyclic
structure of oxazinin A (<b>1</b>) is a unique combination of
benzoxazine, isoquinoline, and a pyran ring. Oxazinin A (<b>1</b>) exhibited antimycobacterial activity and modestly antagonized transient
receptor potential (TRP) channels
Totopotensamides, Polyketide–Cyclic Peptide Hybrids from a Mollusk-Associated Bacterium <i>Streptomyces</i> sp.
Two new compounds, the peptide–polyketide glycoside
totopotensamide A (<b>1</b>) and its aglycone totopotensamide
B (<b>2</b>), were isolated from a <i>Streptomyces</i> sp. cultivated from the gastropod mollusk <i>Lienardia totopotens</i> collected in the Philippines. The compounds contain a previously
undescribed polyketide component, a novel 2,3-diaminobutyric acid-containing
macrolactam, and a new amino acid, 4-chloro-5,7-dihydroxy-6-methylphenylglycine.
The application of Marfey’s method to phenylglycine derivatives
was explored using quantum mechanical calculations and NMR