Covalent linkage of distinct substrate degrons controls assembly and disassembly of DegP proteolytic cages.

Protein quality control requires careful regulation of intracellular proteolysis. For DegP, a periplasmic protease, substrates promote assembly of inactive hexamers into proteolytically active cages with 12, 18, 24, or 30 subunits. Here, we show that sensitive activation and cage assembly require covalent linkage of distinct substrate sequences that affect degradation (degrons). One degron binds the DegP active site, and another degron binds a separate tethering site in PDZ1 in the crystal structure of a substrate-bound DegP dodecamer. FRET experiments demonstrate that active cages assemble rapidly in a reaction that is positively cooperative in substrate concentration, remain stably assembled while uncleaved substrate is present, and dissociate once degradation is complete. Thus, the energy of binding of linked substrate degrons drives assembly of the proteolytic machine responsible for subsequent degradation. Substrate cleavage and depletion results in disassembly, ensuring that DegP is proteolytically active only when sufficient quantities of protein substrates are present.

A dual gene-specific mutator system installs all transition mutations at similar frequencies in vivo.

Targeted in vivo hypermutation accelerates directed evolution of proteins through concurrent DNA diversification and selection. Although systems employing a fusion protein of a nucleobase deaminase and T7 RNA polymerase present gene-specific targeting, their mutational spectra have been limited to exclusive or dominant C:G→T:A mutations. Here we describe eMutaT7transition, a new gene-specific hypermutation system, that installs all transition mutations (C:G→T:A and A:T→G:C) at comparable frequencies. By using two mutator proteins in which two efficient deaminases, PmCDA1 and TadA-8e, are separately fused to T7 RNA polymerase, we obtained similar numbers of C:G→T:A and A:T→G:C substitutions at a sufficiently high frequency (∼6.7 substitutions in 1.3 kb gene during 80-h in vivo mutagenesis). Through eMutaT7transition-mediated TEM-1 evolution for antibiotic resistance, we generated many mutations found in clinical isolates. Overall, with a high mutation frequency and wider mutational spectrum, eMutaT7transition is a potential first-line method for gene-specific in vivo hypermutation.

Development of a genome-targeting mutator for the adaptive evolution of microbial cells.

Methods that can randomly introduce mutations in the microbial genome have been used for classical genetic screening and, more recently, the evolutionary engineering of microbial cells. However, most methods rely on either cell-damaging agents or disruptive mutations of genes that are involved in accurate DNA replication, of which the latter requires prior knowledge of gene functions, and thus, is not easily transferable to other species. In this study, we developed a new mutator for in vivo mutagenesis that can directly modify the genomic DNA. Mutator protein, MutaEco, in which a DNA-modifying enzyme is fused to the α-subunit of Escherichia coli RNA polymerase, increases the mutation rate without compromising the cell viability and accelerates the adaptive evolution of E. coli for stress tolerance and utilization of unconventional carbon sources. This fusion strategy is expected to accommodate diverse DNA-modifying enzymes and may be easily adapted to various bacterial species.

Discovery of Terminal Oxazole-Bearing Natural Products by a Targeted Metabologenomic Approach.

A targeted metabologenomic method was developed to selectively discover terminal oxazole-bearing natural products from bacteria. For this, genes encoding oxazole cyclase, a key enzyme in terminal oxazole biosynthesis, were chosen as the genomic signature to screen bacterial strains that may produce oxazole-bearing compounds. Sixteen strains were identified from the screening of a bacterial DNA library (1,000 strains) using oxazole cyclase gene-targeting polymerase chain reaction (PCR) primers. The PCR amplicon sequences were subjected to phylogenetic analysis and classified into nine clades. 1H-13C coupled-HSQC NMR spectra obtained from the culture extracts of the hit strains enabled the unequivocal detection of the target compounds, including five new oxazole compounds, based on the unique 1JCH values and chemical shifts of oxazole: lenzioxazole (1) possessing an unprecedented cyclopentane, permafroxazole (2) bearing a tetraene conjugated with carboxylic acid, tenebriazine (3) incorporating two modified amino acids, and methyl-oxazolomycins A and B (4 and 5). Tenebriazine displayed inhibitory activity against pathogenic fungi, whereas methyl-oxazolomycins A and B (4 and 5) selectively showed anti-proliferative activity against estrogen receptor-positive breast cancer cells. This metabologenomic method enables the logical and efficient discovery of new microbial natural products with a target structural motif without the need for isotopic labeling.

Exploring the Diverse Landscape of Biaryl-Containing Peptides Generated by Cytochrome P450 Macrocyclases.

Cytochrome P450 enzymes (P450s) catalyze diverse oxidative cross-coupling reactions between aromatic substrates in the natural product biosynthesis. Specifically, P450s install distinct biaryl macrocyclic linkages in three families of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, the chemical diversity of biaryl-containing macrocyclic RiPPs remains largely unexplored. Here, we demonstrate that P450s have the capability to generate diverse biaryl linkages on RiPPs, collectively named "cyptides". Homology-based genome mining for P450 macrocyclases revealed 19 novel groups of homologous biosynthetic gene clusters (BGCs) with distinct aromatic residue patterns in the precursor peptides. Using the P450-modified precursor peptides heterologously coexpressed with corresponding P450s in Escherichia coli, we determined the NMR structures of three novel biaryl-containing peptides─the enzymatic products, roseovertin (1), rubrin (2), and lapparbin (3)─and confirmed the formation of three unprecedented or rare biaryl linkages: Trp C-7'-to-His N-τ in 1, Trp C-7'-to-Tyr C-6 in 2, and Tyr C-6-to-Trp N-1' in 3. Biochemical characterization indicated that certain P450s in these pathways have a relaxed substrate specificity. Overall, our studies suggest that P450 macrocyclases have evolved to create diverse biaryl linkages in RiPPs, promoting the exploration of a broader chemical space for biaryl-containing peptides encoded in bacterial genomes.

Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB.

Graspetides, also known as ω-ester-containing peptides (OEPs), are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) bearing side chain-to-side chain macrolactone or macrolactam linkages. Here, we present the molecular details of precursor peptide recognition by the macrocyclase enzyme PsnB in the biosynthesis of plesiocin, a group 2 graspetide. Biochemical analysis revealed that, in contrast to other RiPPs, the core region of the plesiocin precursor peptide noticeably enhanced the enzyme-precursor interaction via the conserved glutamate residues. We obtained four crystal structures of symmetric or asymmetric PsnB dimers, including those with a bound core peptide and a nucleotide, and suggest that the highly conserved Arg213 at the enzyme active site specifically recognizes a ring-forming acidic residue before phosphorylation. Collectively, this study provides insights into the mechanism underlying substrate recognition in graspetide biosynthesis and lays a foundation for engineering new variants.

Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo.

Various in vivo mutagenesis methods have been developed to facilitate fast and efficient continuous evolution of proteins in cells. However, they either modify the DNA region that does not match the target gene, or suffer from low mutation rates. Here, we report a mutator, eMutaT7 (enhanced MutaT7), with very fast in vivo mutation rate and high gene-specificity in Escherichia coli. eMutaT7, a cytidine deaminase fused to an orthogonal RNA polymerase, can introduce up to ∼4 mutations per 1 kb per day, rivalling the rate in typical in vitro mutagenesis for directed evolution of proteins, and promotes rapid continuous evolution of model proteins for antibiotic resistance and allosteric activation. eMutaT7 provides a very simple and tunable method for continuous directed evolution of proteins, and suggests that the fusion of new DNA-modifying enzymes to the orthogonal RNA polymerase is a promising strategy to explore the expanded sequence space without compromising gene specificity.

Discovery and Biosynthesis of Cihunamides, Macrocyclic Antibacterial RiPPs with a Unique C-N Linkage Formed by CYP450 Catalysis.

Cihunamides A-D (1-4), novel antibacterial RiPPs, were isolated from volcanic-island-derived Streptomyces sp. The structures of 1-4 were elucidated by 1 H, 13 C, and 15 N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C-N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co-expression of the core genes demonstrated the biosynthesis of cihunamides through P450-mediated oxidative Trp-Trp cross-linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including that of tryptorubins, which possess a distinct Trp-Trp linkage. Cihunamides do not display the non-canonical atropisomerism shown in tryptorubins, which are the founding members of the "atropitide" family. Therefore, we propose to use a new RiPP family name, "bitryptides", for cihunamides, tryptorubins, and their congeners, wherein the Trp-Trp linkages define the structural class rather than non-canonical atropisomerism.

Bioinformatic Expansion of Borosins Uncovers Trans-Acting Peptide Backbone N-Methyltransferases in Bacteria.

Backbone N-methylation is one of the prominent peptide modifications that can greatly enhance the pharmacological properties of a peptide. Naturally occurring backbone N-methylated peptides are produced via nonribosomal or ribosomal pathways, the latter of which was only recently identified in the borosin family of ribosomally synthesized and post-translationally modified peptides. Although previous bioinformatic analyses have revealed new putative genes for borosin biosynthesis, the natural scope of structural and biosynthetic diversity of the borosin family has not been thoroughly explored. Here, we report a comprehensive overview of the borosin family of peptide natural products. Using a genome mining approach, we identified more than 1400 new putative biosynthetic gene clusters for borosins and demonstrated that, unlike those previously reported, most of them are found in bacterial genomes and encode a precursor peptide unfused to its cognate methyltransferase enzyme. Biochemical analysis confirmed the backbone N-methylation of the precursor peptide in trans in eight enzyme-precursor pairs and revealed two novel types of enzyme-recognizing sequences in the precursor peptide. This work significantly expands the biosynthetic diversity of borosins and paves the way for the enzymatic production of diverse backbone N-methylated peptides.

Distinct regulatory mechanisms balance DegP proteolysis to maintain cellular fitness during heat stress.

Intracellular proteases combat proteotoxic stress by degrading damaged proteins, but their activity must be carefully controlled to maintain cellular fitness. The activity of Escherichia coli DegP, a highly conserved periplasmic protease, is regulated by substrate-dependent allosteric transformations between inactive and active trimer conformations and by the formation of polyhedral cages that confine the active sites within a proteolytic chamber. Here, we investigate how these distinct control mechanisms contribute to bacterial fitness under heat stress. We found that mutations that increase or decrease the equilibrium population of active DegP trimers reduce high-temperature fitness, that a mutation that blocks cage formation causes a mild fitness decrease, and that combining mutations that stabilize active DegP and block cage formation generates a lethal rogue protease. This lethality is suppressed by an extragenic mutation that prevents covalent attachment of an abundant outer-membrane lipoprotein to peptidoglycan and makes this protein an inhibitor of the rogue protease. Lethality is also suppressed by intragenic mutations that stabilize inactive DegP trimers. In combination, our results suggest that allosteric control of active and inactive conformations is the primary mechanism that regulates DegP proteolysis and fitness, with cage formation providing an additional layer of cellular protection against excessive protease activity.

Verbenalin Reduces Amyloid-Beta Peptide Generation in Cellular and Animal Models of Alzheimer's Disease.

Verbenalin, among the major constituents of Verbena officinalis, has been reported to exhibit sleep-promoting and antioxidant activities. This study demonstrates the effects of verbenalin on amyloid-beta (Aß) peptide generation in Swedish mutant amyloid precursor protein (APP)-overexpressing Neuro2a cells (SweAPP/N2a) and in Alzheimer's disease (AD) animal models. We further performed molecular biological analyses of these in vitro and in vivo models of AD. The effects of verbenalin were assessed based on the expression of factors related to Aß peptide production using Western blotting, enzyme-linked immunosorbent assay, and immunohistochemistry (IHC). The intracellular expression and release of APP protein were both decreased by verbenalin treatment in SweAPP/N2a cells. Thus, the production of Aß peptides was decreased. Compared to those in AD transgenic (Tg) mice, IHC revealed that verbenalin-treated animals showed decreased Aß and tau expression levels in the hippocampus. In addition, verbenalin restored the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus of AD animal models. These findings suggest that verbenalin may decrease Aß formation both in vitro and in vivo. Verbenalin may also help improve the pathological hallmarks of AD.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Introduction of Bifunctionality into the Multidomain Architecture of the ω-Ester-Containing Peptide Plesiocin.

The modular biosynthetic pathway of ribosomally synthesized and post-translationally modified peptides (RiPPs) enhances their engineering potential for exploring new structures and biological functions. The ω-ester-containing peptides (OEPs), a subfamily of RiPPs, have distinct side-to-side ester or amide linkages and frequently present more than one macrocyclic domain in a "beads-on-a-string" structure. In an effort to improve the engineering potential of RiPPs, we present here the idea that the multidomain architecture of an OEP, plesiocin, can be exploited to create a bifunctional modified peptide. Characterization of plesiocin variants revealed that strong chymotrypsin inhibition relies on the bicyclic structure of the domain in which a leucine residue in the hairpin loop functions as a specificity determinant. Four domains of plesiocin promote simultaneous binding of multiple enzymes, where the C-terminal domain binds chymotrypsin most efficiently. Using this information, we successfully engineered a plesiocin variant in which two different domains inhibit chymotrypsin and trypsin. This result suggests that the multidomain architecture of OEPs is a useful platform for engineering multifunctional hybrid RiPPs.

Genome Mining Reveals High Topological Diversity of ω-Ester-Containing Peptides and Divergent Evolution of ATP-Grasp Macrocyclases.

ω-Ester-containing peptides (OEPs) are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) containing intramolecular ω-ester or ω-amide bonds. Although their distinct side-to-side connections may create considerable topological diversity of multicyclic peptides, it is largely unknown how diverse ring patterns have been developed in nature. Here, using genome mining of biosynthetic enzymes of OEPs, we identified genes encoding nine new groups of putative OEPs with novel core consensus sequences, disclosing a total of ∼1500 candidate OEPs in 12 groups. Connectivity analysis revealed that OEPs from three different groups contain novel tricyclic structures, one of which has a distinct biosynthetic pathway where a single ATP-grasp enzyme produces both ω-ester and ω-amide linkages. Analysis of the enzyme cross-reactivity showed that, while enzymes are promiscuous to nonconserved regions of the core peptide, they have high specificity to the cognate core consensus sequence, suggesting that the enzyme-core pair has coevolved to create a unique ring topology within the same group and has sufficiently diversified across different groups. Collectively, our results demonstrate that the diverse ring topologies, in addition to diverse sequences, have been developed in nature with multiple ω-ester or ω-amide linkages in the OEP family of RiPPs.

A Topologically Distinct Modified Peptide with Multiple Bicyclic Core Motifs Expands the Diversity of Microviridin-Like Peptides.

Microviridins are ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain multiple intramolecular ω-ester or ω-amide crosslinks between two side chains in peptides. This type of the side-to-side macrocyclization may generate diverse structures with distinct topology and ring sizes, but the majority of the microviridin-like RiPPs present only a single consensus sequence with a tricyclic architecture. Here, we expanded the natural diversity of the microviridin-like modified peptides by determining the crosslinking connectivity of a new modified peptide, mTgnA and its homologous RiPPs, which we named the thuringinin group. Members of the thuringinin group have core motifs with a distinct consensus sequence, which is transformed to a novel hairpin-like bicyclic structure by the cognate ATP-grasp enzyme. We suggest that the microviridin-like RiPPs naturally have novel sequences and architectures beyond those found in microviridins and comprise a larger RiPP family, termed omega-ester containing peptides (OEPs).

A Small Periplasmic Protein with a Hydrophobic C-Terminal Residue Enhances DegP Proteolysis as a Suicide Activator.

DegP is a highly conserved protease that performs regulated proteolysis to selectively remove misfolded proteins in the periplasm of Escherichia coli Binding of misfolded proteins is known to be the main mechanism of DegP activation, but it is unknown whether any native proteins can alter DegP activity. Here, we show that a small periplasmic protein, YjfN, which is highly upregulated by the Cpx envelope stress response, functions as a "suicide activator" for DegP and promotes efficient degradation of misfolded proteins. YjfN readily binds to and is degraded by DegP, for which a hydrophobic C-terminal residue and transient unfolding of YjfN are critical. YjfN also activates DegP in trans while it is being degraded and accelerates degradation of a denatured outer membrane protein, OmpA, that is not easily recognized by DegP. Although YjfN also prevents OmpA aggregation, the trans-activation effect is mainly responsible for efficient OmpA degradation. Overexpression of YjfN enhances the viability of cells in misfolded protein stress that is induced by the presence of a less-active variant of DegP at high temperature. Collectively, we suggest that YjfN can enhance DegP proteolysis for relieving envelope stresses that may generate toxic misfolded proteins.IMPORTANCE Proper degradation of toxic misfolded proteins is essential for bacterial survival. This function is mainly performed by a highly conserved protease, DegP, in the periplasm of Escherichia coli It is known that binding of misfolded proteins is the main mechanism for activating the DegP protease. Here, we find that a small periplasmic protein, YjfN, can be a substrate and an activator of DegP. It is the first example of a native protein showing an ability to directly alter DegP activity. The YjfN-mediated trans activation of DegP promotes efficient degradation of misfolded proteins. Our results suggest that YjfN is a novel "suicide activator" for DegP that enhances DegP proteolysis under misfolded protein stress.

Identification of Nucleophilic Probes for Protease-Mediated Transpeptidation.

Proteases have evolved to mediate the hydrolysis of peptide bonds but may perform transpeptidation in the presence of a proper nucleophilic molecule that can effectively compete with water to react with the acyl-enzyme intermediate. There have been several examples of protease-mediated transpeptidation, but they are generally inefficient, and little effort has been made to systematically control the transpeptidation activity of other proteases with good nucleophiles. Here, we developed an on-bead screening approach to find a probe that functions efficiently as a nucleophile in the protease-mediated transpeptidation reaction, and we identified good probes for a model protease DegP. These probes were covalently linked to the C-termini of the cleaved peptides in a mild condition and made the selective enrichment of ligated peptides possible. We suggest that good nucleophilic probes can be found for many other proteases that act via acyl-enzyme intermediates, and these probes will help characterize their substrates.

Enzymatic Cross-Linking of Side Chains Generates a Modified Peptide with Four Hairpin-like Bicyclic Repeats.

Macrocyclization of peptides is often employed to generate novel structures and biological activities in the biosynthesis of natural products and drug discovery. The enzymatic cross-linking of two side chains in a peptide via an ester or amide has a high potential for making topologically diverse cyclic peptides but is found with only a single consensus sequence in the microviridin class of natural products. Here, we report that a peptide with a new sequence pattern can be enzymatically cross-linked to make a novel microviridin-like peptide, plesiocin, which contains four repeats of a distinct hairpin-like bicyclic structure and shows strong inhibition of proteases. A single ATP-grasp enzyme binds to a leader peptide, of which only 13 residues are required for binding, and performs eight esterification reactions on the core peptide. We also demonstrate that the combination of tandem mass spectrometry and an ester-specific reaction greatly facilitates the determination of connectivity. We suggest that the enzymatic cross-linking of peptide side chains can generate more diverse structures in nature or by engineering.

Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival.

DegP, a member of the highly conserved HtrA family, performs quality-control degradation of misfolded proteins in the periplasm of gram-negative bacteria and is required for high-temperature survival of Escherichia coli. Substrate binding transforms DegP from an inactive oligomer containing two trimers into active polyhedral cages, typically containing four or eight trimers. Although these observations suggest a causal connection, we show that cage assembly and proteolytic activation can be uncoupled. Indeed, DegP variants that remain trimeric, hexameric, or dodecameric in the presence or absence of substrate still display robust and positively cooperative substrate degradation in vitro and, most importantly, sustain high-temperature bacterial growth as well as the wild-type enzyme. Our results support a model in which substrate binding converts inactive trimers into proteolytically active trimers, and simultaneously leads to cage assembly by enhancing binding of PDZ1 domains in one trimer to PDZ2' domains in neighboring trimers. Thus, both processes depend on substrate binding, but they can be uncoupled without loss of biological function. We discuss potential coupling mechanisms and why cage formation may have evolved if it is not required for DegP proteolysis.

Evolutionary Spread of Distinct O-methyltransferases Guides the Discovery of Unique Isoaspartate-Containing Peptides, Pamtides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways is challenging. Here, it is reported that peptide/protein L-aspartyl O-methyltransferases (PAMTs) present in several RiPP subclasses are highly homologous. Importantly, it is discovered that the apparent evolutionary transmission of the PAMT gene to unrelated RiPP subclasses can serve as a basis to identify a novel RiPP subclass. Biochemical and structural analyses suggest that homologous PAMTs convert aspartate to isoaspartate via aspartyl-O-methyl ester and aspartimide intermediates, and often require cyclic or hairpin-like structures for modification. By conducting homology-based bioinformatic analysis of PAMTs, over 2,800 biosynthetic gene clusters (BGCs) are identified for known RiPP subclasses in which PAMTs install a secondary modification, and over 1,500 BGCs where PAMTs function as a primary modification enzyme, thereby defining a new RiPP subclass, named pamtides. The results suggest that the genome mining of proteins with secondary biosynthetic roles can be an effective strategy for discovering novel biosynthetic pathways of RiPPs through the principle of "guilt by association".