11 research outputs found
Post-translational Claisen Condensation and Decarboxylation en Route to the Bicyclic Core of Pantocin A
Pantocin A (PA) is a member of the growing family of ribosomally encoded and post-translationally modified peptide natural products (RiPPs). PA is much smaller than most known RiPPs, a tripeptide with a tight bicyclic core that appears to be cleaved from the middle of a larger 30-residue precursor peptide. We show here that the enzyme PaaA catalyzes the double dehydration and decarboxylation of two glutamic acid residues in the 30-residue precursor PaaP. Further truncates of PaaP leader and follower peptide sequences demonstrate the different impacts of these two regions on PaaA-mediated tailoring and delineate an essential role for the follower sequence in the decarboxylation step. The crystal structure of apo PaaA is reported, allowing identification of structural features that set PaaA apart from other homologous enzymes that typically do not catalyze such extended post-translational chemistry. Together, these data reveal how additional chemistry can be extracted from a ubiquitous enzyme family toward ribosomally derived peptide natural product biosynthesis and suggest that more examples of such enzymes likely exist in untapped genomic space
Discovery of a Previously Unrecognized Ribonuclease from <i>Escherichia coli</i> That Hydrolyzes 5′-Phosphorylated Fragments of RNA
TrpH or YciV (locus tag b1266) from <i>Escherichia coli</i> is annotated as a protein of unknown function
that belongs to the
polymerase and histidinol phosphatase (PHP) family of proteins in
the UniProt and NCBI databases. Enzymes from the PHP family have been
shown to hydrolyze organophosphoesters using divalent metal ion cofactors
at the active site. We found that TrpH is capable of hydrolyzing the
3′-phosphate from 3′,5′-bis-phosphonucleotides.
The enzyme will also sequentially hydrolyze 5′-phosphomononucleotides
from 5′-phosphorylated RNA and DNA oligonucleotides, with no
specificity toward the identity of the nucleotide base. The enzyme
will not hydrolyze RNA or DNA oligonucleotides that are unphosphorylated
at the 5′-end of the substrate, but it makes no difference
whether the 3′-end of the oligonucleotide is phosphorylated.
These results are consistent with the sequential hydrolysis of 5′-phosphorylated
mononucleotides from oligonucleotides in the 5′ → 3′
direction. The catalytic efficiencies for hydrolysis of 3′,5′-pAp,
pÂ(Ap)ÂA, pÂ(Ap)<sub>4</sub>A, and pÂ(dAp)<sub>4</sub>dA were determined
to be 1.8 × 10<sup>5</sup>, 9.0 × 10<sup>4</sup>, 4.6 ×
10<sup>4</sup>, and 2.9 × 10<sup>3</sup> M<sup>–1</sup> s<sup>–1</sup>, respectively. TrpH was found to be more efficient
at hydrolyzing RNA oligonucleotides than DNA oligonucleotides. This
enzyme can also hydrolyze annealed DNA duplexes, albeit at a catalytic
efficiency approximately 10-fold lower than that of the corresponding
single-stranded oligonucleotides. TrpH is the first enzyme from <i>E. coli</i> that has been found to possess 5′ →
3′ exoribonuclease activity. We propose to name this enzyme
RNase AM
Post-translational Claisen Condensation and Decarboxylation en Route to the Bicyclic Core of Pantocin A
Pantocin A (PA) is a member of the growing family of ribosomally encoded and post-translationally modified peptide natural products (RiPPs). PA is much smaller than most known RiPPs, a tripeptide with a tight bicyclic core that appears to be cleaved from the middle of a larger 30-residue precursor peptide. We show here that the enzyme PaaA catalyzes the double dehydration and decarboxylation of two glutamic acid residues in the 30-residue precursor PaaP. Further truncates of PaaP leader and follower peptide sequences demonstrate the different impacts of these two regions on PaaA-mediated tailoring and delineate an essential role for the follower sequence in the decarboxylation step. The crystal structure of apo PaaA is reported, allowing identification of structural features that set PaaA apart from other homologous enzymes that typically do not catalyze such extended post-translational chemistry. Together, these data reveal how additional chemistry can be extracted from a ubiquitous enzyme family toward ribosomally derived peptide natural product biosynthesis and suggest that more examples of such enzymes likely exist in untapped genomic space
Discovery of a Cyclic Phosphodiesterase That Catalyzes the Sequential Hydrolysis of Both Ester Bonds to Phosphorus
The
bacterial C–P lyase pathway is responsible for the metabolism
of unactivated organophosphonates under conditions of phosphate starvation.
The cleavage of the C–P bond within ribose-1-methylphosphonate-5-phosphate
to form methane and 5-phospho-ribose-1,2-cyclic phosphate (PRcP) is
catalyzed by the radical SAM enzyme PhnJ. In <i>Escherichia coli</i> the cyclic phosphate product is hydrolyzed to ribose-1,5-bisphosphate
by PhnP. In this study, we describe the discovery and characterization
of an enzyme that can hydrolyze a cyclic phosphodiester directly to
a vicinal diol and inorganic phosphate. With PRcP, this enzyme hydrolyzes
the phosphate ester at carbon-1 of the ribose moiety to form ribose-2,5-bisphosphate,
and then this intermediate is hydrolyzed to ribose-5-phosphate and
inorganic phosphate. Ribose-1,5-bisphosphate is neither an intermediate
nor a substrate for this enzyme. Orthologues of this enzyme are found
in the human pathogens <i>Clostridium difficile</i> and <i>Eggerthella lenta</i>. We propose that this enzyme be called
cyclic phosphate dihydrolase (cPDH) and be designated as PhnPP
Prospecting for Unannotated Enzymes: Discovery of a 3′,5′-Nucleotide Bisphosphate Phosphatase within the Amidohydrolase Superfamily
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Structural and Mechanistic Characterization of l‑Histidinol Phosphate Phosphatase from the Polymerase and Histidinol Phosphatase Family of Proteins
l-Histidinol phosphate phosphatase (HPP) catalyzes
the
hydrolysis of l-histidinol phosphate to l-histidinol
and inorganic phosphate, the penultimate step in the biosynthesis
of l-histidine. HPP from the polymerase and histidinol phosphatase
(PHP) family of proteins possesses a trinuclear active site and a
distorted (β/α)<sub>7</sub>-barrel protein fold. This
group of enzymes is closely related to the amidohydrolase superfamily
of enzymes. The mechanism of phosphomonoester bond hydrolysis by the
PHP family of HPP enzymes was addressed. Recombinant HPP from <i>Lactococcus lactis</i> subsp. <i>lactis</i> that was
expressed in <i>Escherichia coli</i> contained a mixture
of iron and zinc in the active site and had a catalytic efficiency
of ∼10<sup>3</sup> M<sup>–1</sup> s<sup>–1</sup>. Expression of the protein under iron-free conditions resulted in
the production of an enzyme with a 2 order of magnitude improvement
in catalytic efficiency and a mixture of zinc and manganese in the
active site. Solvent isotope and viscosity effects demonstrated that
proton transfer steps and product dissociation steps are not rate-limiting.
X-ray structures of HPP were determined with sulfate, l-histidinol
phosphate, and a complex of l-histidinol and arsenate bound
in the active site. These crystal structures and the catalytic properties
of variants were used to identify the structural elements required
for catalysis and substrate recognition by the HPP family of enzymes
within the amidohydrolase superfamily
P450-Mediated Non-natural Cyclopropanation of Dehydroalanine-Containing Thiopeptides
Thiopeptides
are a growing class of ribosomally synthesized and
post-translationally modified peptide (RiPP) natural products. Many
biosynthetic enzymes for RiPPs, especially thiopeptides, are promiscuous
and can accept a wide range of peptide substrates with different amino
acid sequences; thus, these enzymes have been used as tools to generate
new natural product derivatives. Here, we explore an alternative route
to molecular complexity by engineering thiopeptide tailoring enzymes
to do new or non-native chemistry. We explore cytochrome P450 enzymes
as biocatalysts for cyclopropanation of dehydroalanines, chemical
motifs found widely in thiopeptides and other RiPP-based natural products.
We find that P450<sub>TbtJ1</sub> and P450<sub>TbtJ2</sub> selectively
cyclopropanate dehydroalanines in a number of complex thiopeptide-based
substrates and convert them into 1-amino-2-cyclopropane carboxylic
acids (ACCAs), which are important pharmacophores. This chemistry
takes advantage of the innate affinity of these biosynthetic enzymes
for their substrates and enables incorporation of new pharmacophores
into thiopeptide architectures. This work also presents a strategy
for diversification of natural products through rationally repurposing
biosynthetic enzymes as non-natural biocatalysts
Prospecting for Unannotated Enzymes: Discovery of a 3′,5′-Nucleotide Bisphosphate Phosphatase within the Amidohydrolase Superfamily
In
bacteria, 3′,5′-adenosine bisphosphate (pAp) is
generated from 3′-phosphoadenosine 5′-phosphosulfate
in the sulfate assimilation pathway, and from coenzyme A by the transfer
of the phosphopantetheine group to the acyl-carrier protein. pAp is
subsequently hydrolyzed to 5′-AMP and orthophosphate, and this
reaction has been shown to be important for superoxide stress tolerance.
Herein, we report the discovery of the first instance of an enzyme
from the amidohydrolase superfamily that is capable of hydrolyzing
pAp. Crystal structures of Cv1693 from <i>Chromobacterium violaceum</i> have been determined to a resolution of 1.9 Ã… with AMP and
orthophosphate bound in the active site. The enzyme has a trinuclear
metal center in the active site with three Mn<sup>2+</sup> ions. This
enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613
from the polymerase and histidinol phosphatase family of enzymes.
The values of <i>k</i><sub>cat</sub> and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for the hydrolysis
of pAp are 22 s<sup>–1</sup> and 1.4 × 10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup>, respectively. The enzyme is
promiscuous and is able to hydrolyze other 3′,5′-bisphosphonucleotides
(pGp, pCp, pUp, and pIp) and 2′-deoxynucleotides with comparable
catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides
(pdA)<sub>5</sub>, albeit at rates much lower than that of pAp. Enzymes
from two other enzyme families have previously been found to hydrolyze
pAp at physiologically significant rates. These enzymes include CysQ
from <i>Escherichia coli</i> (cog1218) and YtqI/NrnA from <i>Bacillus subtilis</i> (cog0618). Identification of the functional
homologues to the experimentally verified pAp phosphatases from cog0613,
cog1218, and cog0618 suggests that there is relatively little overlap
of enzymes with this function in sequenced bacterial genomes