In recent years, the number of known peptide natural products that are synthesized via the ribosomal pathway has rapidly grown. Taking advantage of sequence homology among genes encoding precursor peptides or biosynthetic proteins, in silico mining of genomes combined with molecular biology approaches has guided the discovery of a large number of new ribosomal natural products, including lantipeptides, cyanobactins, linear thiazole/oxazole-containing peptides, microviridins, lasso peptides, amatoxins, cyclotides, and conopeptides. In this review, we describe the strategies used for the identification of these ribosomally-synthesized and posttranslationally modified peptides (RiPPs) and the structures of newly identified compounds. The increasing number of chemical entities and their remarkable structural and functional diversity may lead to novel pharmaceutical applications.
Lantibiotics are a family of antibacterial peptide natural products characterized by the posttranslational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single stereochemical configuration for each of these crosslinks was known in Nature. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing crosslinks with non-native stereochemical configuration. Biological evaluation revealed that these alterations abolished antibacterial activity in all analogues, revealing the critical importance of the enzymatically-installed stereochemistry for the biological activity of lacticin 481.
The avalanche of genomic information in the past decade has revealed that natural product biosynthesis using the ribosomal machinery is much more widespread than originally anticipated. Nearly all of these compounds are crafted through posttranslational modifications of a larger precursor peptide that often contains the marching orders for the biosynthetic enzymes. We review here the available information for how the peptide sequences in the precursors govern the posttranslational tailoring processes for several classes of natural products. In addition, we highlight the great potential these leader peptide directed biosynthetic systems offer for engineering conformationally restrained and pharmacophore-rich products with structural diversity that greatly expands the proteinogenic repertoire.
We report the heterologous production of Ala(0)actagardine in E. coli by co-expression of the substrate peptide GarA and its modification enzymes GarM and GarO. The activity of GarO, a luciferase-like monooxygenase that introduces the unique sulfoxide group of actagardine, was also investigated in vitro.
The enterococcal cytolysin is a two-component lantibiotic of unknown structure with hemolytic activity that is important for virulence. We prepared cytolysin by co-expression of each precursor peptide with the synthetase CylM in E. coli, and characterized its structure. Surprisingly, cytolysin is the first example of a lantibiotic containing lanthionine and methyllanthionine structures with different stereochemistries in the same peptide, which is determined by the sequence of the substrate peptide.
Phosphorylation is an abundant post-translational modification involved in a myriad of eukaryotic cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pS) and O-phosphothreonine (pT) residues, either in vitro or in vivo.
The posttranslational methylation of N-terminal α-amino groups (α-N-methylation) is a ubiquitous reaction found in all domains of life. Although this modification usually occurs on protein substrates, recent studies have shown that it also takes place on ribosomally synthesized natural products. Here we report an investigation of the bacterial α-N-methyltransferase CypM involved in the biosynthesis of the peptide antibiotic cypemycin. We demonstrate that CypM has low substrate selectivity and methylates a variety of oligopeptides, cyclic peptides such as nisin and haloduracin, and the ε-amino group of lysine. Hence it may have potential for enzyme engineering and combinatorial biosynthesis. Bayesian phylogenetic inference of bacterial α-N-methyltransferases suggests that they have not evolved as a specific group based on the chemical transformations they catalyze, but that they have been acquired from various other methyltransferase classes during evolution.
α-N-methyltransferase; peptide antibiotic; lantibiotic; catalytic promiscuity; evolution
Relative to the atmosphere, much of the aerobic ocean is supersaturated with methane; however, the source of this important greenhouse gas remains enigmatic. Catabolism of methylphosphonic acid by phosphorus-starved marine microbes, with concomitant release of methane, has been suggested to explain this phenomenon, yet methylphosphonate is not a known natural product, nor has it been detected in natural systems. Further, its synthesis from known natural products would require unknown biochemistry. Here we show that the marine archaeon Nitrosopumilus maritimus encodes a pathway for methylphosphonate biosynthesis and that it produces cell-associated methylphosphonate esters. The abundance of a key gene in this pathway in metagenomic datasets suggests that methylphosphonate biosynthesis is relatively common in marine microbes, providing a plausible explanation for the methane paradox.
Fosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.
catabolism; enzyme catalysis; lyases; phosphonates; S-adenosylmethionine enzymes
Mutants of the highly promiscuous lantipeptide synthetase ProcM phosphorylate a wide range of peptides attached to ProcA leader peptides, both in vitro and in E. coli. As such, the ProcM mutants are useful enzymes for the enzymatic preparation of phosphorylated peptides.
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.
Disulfide bonds are essential for the structural stability and biological activity of many bioactive peptides. However, these bonds are labile to reducing agents, which can limit the therapeutic utility of such peptides. Substitution of a disulfide bond with a reduction-resistant cystathionine bridge is an attractive means of improving stability while imposing minimal structural perturbation to the peptide. We have applied this approach to the therapeutic complement inhibitor compstatin, a disulfide-containing peptide currently in clinical trials for age-related macular degeneration, in an effort to maintain its potent activity while improving its biological stability. Thioether-containing compstatin analogues were produced via solid-phase peptide synthesis utilizing orthogonally-protected cystathionine amino acid building blocks and solid-supported peptide cyclization. Overall, the affinity of these analogues for their biological target and potent inhibition of complement activation were largely maintained when compared to those of the parent disulfide-containing peptides. Thus, the improved stability to reduction conferred by the thioether bond makes this new class of compstatin peptides a promising alternative for therapeutic applications. Additionally, the versatility of this synthesis allows for exploration of this disulfide-to-thioether substitution in a variety of other therapeutic peptides.
Cyclooxygenase catalysis by prostaglandin H synthase (PGHS) is thought to involve a multistep mechanism with several radical intermediates. The proposed mechanism begins with transfer of the C13 pro-(S) hydrogen atom from the substrate arachidonic acid (AA) to the Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. The importance of the hydrogen-transfer step to controlling the overall kinetics of cyclooxygenase catalysis has not been directly examined. We quantified the non-competitive primary kinetic isotope effect (KIE) for both PGHS-1 and -2 using unlabeled AA and several deuterated AAs, including 13-pro-(S) d-AA, 13,13-d2-AA and 10, 10, 13,13-d4-AA. The primary KIE for steady-state cyclooxygenase catalysis, Dkcat, ranged between 1.8 and 2.3 in oxygen electrode measurements. The intrinsic KIE of AA radical formation by C13 pro-(S) hydrogen abstraction in PGHS-1 was estimated to be 1.9–2.3 using rapid freeze-quench EPR kinetic analysis of anaerobic reactions and computer modeling to a mechanism that includes slow formation of a pentadienyl AA radical and rapid equilibration of the AA radical with a tyrosyl radical, NS1c. The observation of similar values for steady-state and pre-steady state KIEs suggests that hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. The large difference of the observed KIE from that of lipoxygenase indicates very different mechanism of hydrogen transfer.
Incubation of prostaglandin H synthase-1 (PGHS-1) under anaerobic conditions with peroxide and arachidonic acid leads to two major radical species: a pentadienyl radical and a radical with a narrow EPR spectrum. The proportions of the two radicals are sensitive to temperature, favoring the narrow radical species at 22 °C. The EPR characteristics of this latter radical are somewhat similar to the previously reported narrow-singlet tyrosine radical NS1a and are insensitive to deuterium labeling of AA. To probe the origin and structure of this radical, we combined EPR analysis with nitric oxide (NO) trapping of tyrosine and substrate derived radicals for both PGHS-1 and -2. Formation of 3-nitrotyrosine in the proteins was analyzed by immunoblotting, whereas NO adducts to AA and AA metabolites were analyzed by mass spectrometry and by chromatography of 14C-labeled products. The results indicate that both nitrated tyrosine residues and NO-AA adducts formed upon NO trapping. The NO-AA adduct was predominantly an oxime at C11 of AA with three conjugated double bonds, as indicated by absorption at 275 nm and by mass spectral analysis. This adduct amounted to 10% and 20% of the heme concentration of PGHS-1 and -2, respectively. For PGHS-1, the yield of NO-AA adduct matched the yield of the narrow radical signal obtained in parallel EPR experiments. High frequency EPR characterization of this narrow radical, reported in an accompanying paper, supports assignment to a new tyrosyl radical, NS1c, rather than an AA-based radical. To reconcile the results from EPR and NO-trapping studies, we propose that the NS1c is in equilibrium with an AA pentadienyl radical, and that the latter reacts preferentially with NO.
Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O2. The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.
PGHS radical; EPR; High-field EPR; Radical dynamics
Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides. The recently discovered lantibiotic epilancin 15X produced by Staphylococcus epidermidis 15X154 contains an unusual N-terminal lactate group. To understand its biosynthesis, the epilancin 15X biosynthetic gene cluster was identified. The N-terminal lactate is produced by dehydration of a Ser residue in the first position of the core peptide by ElxB, followed by proteolytic removal of the leader peptide by ElxP, and hydrolysis of the resulting new N-terminal dehydroalanine. The pyruvate group thus formed is reduced to lactate by an NADPH dependent oxidoreductase designated ElxO. The enzymatic activity of ElxB, ElxP, and ElxO were investigated in vitro or in vivo and the importance of the N-terminal modification for peptide stability against bacterial aminopeptidases was assessed.
The metabolic processing of dehydrophos, a broad-spectrum peptide antibiotic containing an unusual vinyl-phosphonate moiety, was examined by using a panel of Salmonella enterica mutants deficient in peptide uptake and catabolism. Dehydrophos bioactivity is lost in opp tpp double mutants, demonstrating a requirement for uptake via nonspecific oligopeptide permeases. Dehydrophos bioactivity is also abolished in a quadruple Salmonella mutant lacking the genes encoding peptidases A, B, D, and N, showing that hydrolysis of the peptide bond is required for activity. 31P nuclear magnetic resonance spectroscopy was used to assess the fate of dehydrophos following in vitro digestion of the antibiotic with purified PepA. The results suggest that the initial product of peptidase processing is 1-aminovinyl-phosphonate O-methyl ester. This phosphonate analogue of dehydroalanine undergoes rearrangement to the more stable imine, followed by spontaneous hydrolysis to yield O-methyl-acetylphosphonate, a structural analogue of pyruvate. This compound is a known inhibitor of pyruvate dehydrogenase and pyruvate oxidase and is probably the active species responsible for dehydrophos bioactivity.
Sublancin is shown to be an S-linked glycopeptide containing a glucose attached to a Cys residue, establishing a new post-translational modification. The activity of the S-glycosyl transferase was reconstituted in vitro and the enzyme is shown to have relaxed substrate specificity allowing the preparation of analogs of sublancin. Glycosylation is essential for its antimicrobial activity.
The interactions of the axial ligands with copper are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of blue copper sites with a weak axial ligand to green copper sites containing a medium strength axial ligand has been demonstrated in cupredoxins, converting blue copper sites to a red copper site with a strong axial ligand has not been reported. Here we show that replacing Met121 in azurin from Pseudomonas aeruginosa with Cys caused an increased ratio (RL) of absorption at 447 nm over that at 621 nm. While no axial Cu-S(Cys121) interaction in Met121Cys was detectable by the extended x-ray absorption fine structure (EXAFS) at pH 5, similar to what was observed in WT azurin with Met121 as the axial ligand, the Cu-S(Cys121) interaction at 2.74 Å is clearly visible at higher pH. Despite the higher RL and stronger axial Cys121 interaction with Cu(II) ion, the Met121Cys variant remains largely a type 1 copper protein at low pH (with hyperfine coupling constant A|| = 54 × 10−4 cm−1 at pH 4 and 5), or distorted type 1 or green copper protein at high pH (A|| = 87 × 10−4 cm−1 at pH 8 and 9), attributable to the relatively long distance between the axial ligand and copper and the constraint placed by the protein scaffold. To shorten the distance between axial ligand and copper, we replaced Met121 with the nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (RL= 1.5, and A|| = 180 × 10−4 cm−1) and Cu-S(Cys) distance (2.22 Å) are very similar to those of the red copper protein nitrosocyanin. Replacing Met121 with Cys resulted in lowering of the reduction potential from 222 mV in the native azurin to 95 ± 3 mV for Met121Cys azurin and 113 ± 6 mV for Met121Hcy at pH 7. The results strongly support the “coupled distortion” model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions.
Recent years have seen a rapid increase in the mechanistic and structural information on enzymes that are involved in the biosynthesis and breakdown of naturally occurring phosphonates. This review focuses on these recent developments with an emphasis on those enzymes that have been characterized crystallographically in the past five years, including proteins involved in the biosynthesis of phosphinothricin, fosfomycin, and dehydrophos and proteins involved in resistance mechanisms.
antibiotics; phosphonates; X-ray crystallography; methyl transferase; dioxygenase; epoxidase; resistance
We report a photolabile linker compatible with Fmoc solid phase peptide synthesis and Cu(I)-catalyzed alkyne–azide cycloaddition that allows photochemical cleavage to afford a C-terminal peptide fragment with a native amino terminus.
Synthetic derivatives of the phosphonate antibiotic dehydrophos were tested for antimicrobial activity. Both the phosphonate monomethyl ester and the vinyl phosphonate moiety proved to be important for bacteriocidal activity of the natural product.
A growing number of non-heme-iron oxygenases and oxidases catalyze reactions for which the well-established mechanistic paradigm involving a single C-H-bond cleaving intermediate of the Fe(IV)-oxo (ferryl) type  is insufficient to explain the chemistry. It is becoming clear that, in several of these cases, Fe(III)-superoxide complexes formed by simple addition of O2 to the reduced [Fe(II)] cofactor initiate substrate oxidation by abstracting hydrogen . This substrate-oxidizing entry route into high-valent-iron intermediates makes possible an array of complex and elegant oxidation reactions without consumption of valuable reducing equivalents. Examples of this novel mechanistic strategy are discussed with the goal of bringing forth unifying principles.
Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of Streptomyces lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments.
phosphonate; antibiotic; Streptomyces; biosynthetic; gene cluster
During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.
Using an in vitro model of infection, we evaluated the influence of the germination state of B. anthracis spores, as controlled by defined culture conditions, on the outcome of infection. Spores prepared from B. anthracis Sterne 7702 germinated in a variety of common cell culture media supplemented with fetal bovine serum (FBS) while, in the absence of FBS, germination was strictly dependent on medium composition. RAW264.7 macrophage-like cells internalized spores to the same extent in either germinating or non-germinating media. However, significantly more viable, intracellular B. anthracis were recovered from cells infected under non-germinating conditions compared to germinating conditions. At the same time, RAW264.7 cells demonstrated a significant loss in viability when infected under non-germinating conditions.
These results suggest that the outcome of host cell infection is sensitive to the germination state of spores at the time of uptake. Moreover, this study demonstrates the efficacy of studying B. anthracis spore infection of host cells within a defined, non-germinating, in vitro environment.