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, p­(Ap)­A, p­(Ap)₄A, and p­(dAp)₄dA were determined to be 1.8 × 10⁵, 9.0 × 10⁴, 4.6 × 10⁴, and 2.9 × 10³ M^–1 s^–1, 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)₇-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 ∼10³ M^–1 s^–1. 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_TbtJ1 and P450_TbtJ2 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 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn²⁺ 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>_cat and <i>k</i>_cat/<i>K</i>_m for the hydrolysis of pAp are 22 s^–1 and 1.4 × 10⁶ M^–1 s^–1, 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)₅, 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