Macrocyclic Oxindole Peptide Epoxyketones-A Comparative Study of Macrocyclic Inhibitors of the 20S Proteasome.

Peptide macrocycles have recently gained attention as protease inhibitors due to their metabolic stability and specificity. However, the development of peptide macrocycles with improved binding potency has so far been challenging. Here we present macrocyclic peptides derived from the clinically applied proteasome inhibitor carfilzomib with an oxindole group that mimics the natural product TMC-95A. Fluorescence kinetic activity assays reveal a high potency of the oxindole group (IC50 = 0.19 µM) compared with agents lacking this motif. X-ray structures of the ligands with the ß5-subunit of the yeast 20S proteasome illustrate that the installed macrocycle forces strong hydrogen bonding of the oxindole group with ß5-Gly23NH. Thus, the binding of our designed oxindole epoxyketones is entropically and enthalpically favored in contrast to more flexible proteasome inhibitors such as carfilzomib.

Polyketide Trimming Shapes Dihydroxynaphthalene-Melanin and Anthraquinone Pigments.

Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold. Here, multiple crystal structures of PlAntI in various conformations and with bound substrate surrogates or inhibitors are reported. Structure-based mutagenesis and activity assays provide experimental insights into the three sequential reaction steps to yield the natural product AQ-256. For comparison, a series of ligand-complex structures of two functionally related hydrolases involved in the biosynthesis of 1,8-dihydroxynaphthalene-melanin in pathogenic fungi is determined. These data provide fundamental insights into the mechanism of polyketide trimming that shapes pigments in pro- and eukaryotes.

Evolution-inspired engineering of nonribosomal peptide synthetases.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

Structure of Staphylococcus aureus ClpP Bound to the Covalent Active-Site Inhibitor Cystargolide A.

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural ß-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by ß-lactones, the predominant class of ClpP inhibitors.

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

Optical Control of Proteasomal Protein Degradation with a Photoswitchable Lipopeptide.

Photolipids have emerged as attractive tools for the optical control of lipid functions. They often contain an azobenzene photoswitch that imparts a cis double-bond upon irradiation. Herein, we present the application of photoswitching to a lipidated natural product, the potent proteasome inhibitor cepafungin I. Several azobenzene-containing lipids were attached to the cyclopeptide core, yielding photoswitchable derivatives. Most notably, PhotoCep4 exhibited a 10-fold higher cellular potency in its light-induced cis-form, matching the potency of natural cepafungin I. The length of the photolipid tail and distal positioning of the azobenzene photoswitch with respect to the macrocycle is critical for this activity. In a proteome-wide experiment, light-triggered PhotoCep4 modulation showed high overlap with constitutively active cepafungin I. The mode of action was studied using crystallography and revealed an identical binding of the cyclopeptide in comparison to cepafungin I, suggesting that differences in their cellular activity originate from switching the tail structure. The photopharmacological approach described herein could be applicable to many other natural products as lipid conjugation is common and often necessary for potent activity. Such lipids are often introduced late in synthetic routes, enabling facile chemical modifications.

Meet the authors: Dr. Eva Huber, Dr. Michael Groll, and Dr. Eric Ortlund.

In this meet-the-author Q&A, Cell Press Community Review editor Leslie Nitsche speaks to Eric Ortlund as well as Eva Huber and Michael Groll from the Groll group about their recent Structure papers and their experiences publishing via Cell Press Community Review.

Metal-dependent enzyme symmetry guides the biosynthetic flux of terpene precursors.

Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential.

A Chemical Proteomic Strategy Reveals Inhibitors of Lipoate Salvage in Bacteria and Parasites.

The development of novel anti-infectives requires unprecedented strategies targeting pathways which are solely present in pathogens but absent in humans. Following this principle, we developed inhibitors of lipoic acid (LA) salvage, a crucial pathway for the survival of LA auxotrophic bacteria and parasites but non-essential in human cells. An LA-based probe was selectively transferred onto substrate proteins via lipoate protein ligase (LPL) in intact cells, and their binding sites were determined by mass spectrometry. Probe labeling served as a proxy of LPL activity, enabling in situ screenings for cell-permeable LPL inhibitors. Profiling a focused compound library revealed two substrate analogs (LAMe and C3) as inhibitors, which were further validated by binding studies and co-crystallography. Importantly, LAMe exhibited low toxicity in human cells and achieved killing of Plasmodium falciparum in erythrocytes with an EC50 value of 15 µM, making it the most effective LPL inhibitor reported to date.

Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b.

Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.

A set of closely related methyltransferases for site-specific tailoring of anthraquinone pigments.

Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O-methyltransferases encoded in the genome of P. luminescens. Activity assays in vitro and deletion experiments in vivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found in vitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation.

Heterocomplex structure of a polyketide synthase component involved in modular backbone halogenation.

Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid biosynthesis. The cytotoxic oocydin-type polyketides contain a vinyl chloride moiety introduced during polyketide chain elongation. Required for modular polyketide backbone halogenation are a non-heme iron and ɑ-ketoglutarate-dependent halogenase OocP and OocQ lacking characterized homologs. This work provides structural insights into these unusual PKS components and their interactions via a high-resolution X-ray crystallography structure of the heterocomplex. By mapping the protein-protein interactions and comparison with structures of similar halogenases, we illustrate the potential of this heterodimer complex as a replacement for the conserved homodimeric structure of homologous enzymes. The OocPQ protein pair has thus evolved as a means of stabilizing the halogenase and facilitating chemical transformations with great synthetic utility.

Structural and Biochemical Insights into Post-Translational Arginine-to-Ornithine Peptide Modifications by an Atypical Arginase.

Landornamide A is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product with antiviral activity. Its biosynthetic gene cluster encodes─among other maturases─the peptide arginase OspR, which converts arginine to ornithine units in an unusual post-translational modification. Peptide arginases are a recently discovered RiPP maturase family with few characterized representatives. They show little sequence similarity to conventional arginases, a well-characterized enzyme family catalyzing the hydrolysis of free arginine to ornithine and urea. Peptide arginases are highly promiscuous and accept a variety of substrate sequences. The molecular basis for binding the large peptide substrate and for the high promiscuity of peptide arginases remains unclear. Here, we report the first crystal structure of a peptide arginase at a resolution of 2.6 Å. The three-dimensional structure reveals common features and differences between conventional arginases and the peptide arginase: the binuclear metal cluster and the active-site environment strongly resemble each other, while the quaternary structures diverge. Kinetic analyses of OspR with various substrates provide new insights into the order of biosynthetic reactions during the post-translational maturation of landornamide A. These results provide the basis for pathway engineering to generate derivatives of landornamide A and for the general application of peptide arginases as biosynthetic tools for peptide engineering.

Peroxy Intermediate Drives Carbon Bond Activation in the Dioxygenase AsqJ.

Dioxygenases catalyze stereoselective oxygen atom transfer in metabolic pathways of biological, industrial, and pharmaceutical importance, but their precise chemical principles remain controversial. The α-ketoglutarate (αKG)-dependent dioxygenase AsqJ synthesizes biomedically active quinolone alkaloids via desaturation and subsequent epoxidation of a carbon-carbon bond in the cyclopeptin substrate. Here, we combine high-resolution X-ray crystallography with enzyme engineering, quantum-classical (QM/MM) simulations, and biochemical assays to describe a peroxidic intermediate that bridges the substrate and active site metal ion in AsqJ. Homolytic cleavage of this moiety during substrate epoxidation generates an activated high-valent ferryl (FeIV = O) species that mediates the next catalytic cycle, possibly without the consumption of the metabolically valuable αKG cosubstrate. Our combined findings provide an important understanding of chemical bond activation principles in complex enzymatic reaction networks and molecular mechanisms of dioxygenases.

Biochemical and Structural Characterization of Thermostable GH159 Glycoside Hydrolases Exhibiting α-L-Arabinofuranosidase Activity.

Functional, biochemical, and preliminary structural properties are reported for three glycoside hydrolases of the recently described glycoside hydrolase (GH) family 159. The genes were cloned from the genomic sequences of different Caldicellulosiruptor strains. This study extends the spectrum of functions of GH159 enzymes. The only activity previously reported for GH159 was hydrolytic activity on ß-galactofuranosides. Activity screening using a set of para-nitrophenyl (pNP) glycosides suggested additional arabinosidase activity on substrates with arabinosyl residues, which has not been previously reported for members of GH159. Even though the thermophilic enzymes investigated-Cs_Gaf159A, Ch_Gaf159A, and Ck_Gaf159A-cleaved pNP-α-l-arabinofuranoside, they were only weakly active on arabinogalactan, and they did not cleave arabinose from arabinan, arabinoxylan, or gum arabic. However, the enzymes were able to hydrolyze the α-1,3-linkage in different arabinoxylan-derived oligosaccharides (AXOS) with arabinosylated xylose at the non-reducing end (A3X, A2,3XX), suggesting their role in the intracellular hydrolysis of oligosaccharides. Crystallization and structural analysis of the apo form of one of the Caldicellulosiruptor enzymes, Ch_Gaf159A, enabled the elucidation of the first 3D structure of a GH159 member. This work revealed a five-bladed ß-propeller structure for GH159 enzymes. The 3D structure and its substrate-binding pocket also provides an explanation at the molecular level for the observed exo-activity of the enzyme. Furthermore, the structural data enabled the prediction of the catalytic amino acids. This was supported by the complete inactivation by mutation of residues D19, D142, and E190 of Ch_Gaf159A.

A Specialized Polythioamide-Binding Protein Confers Antibiotic Self-Resistance in Anaerobic Bacteria.

Understanding antibiotic resistance mechanisms is central to the development of anti-infective therapies and genomics-based drug discovery. Yet, many knowledge gaps remain regarding the resistance strategies employed against novel types of antibiotics from less-explored producers such as anaerobic bacteria, among them the Clostridia. Through the use of genome editing and functional assays, we found that CtaZ confers self-resistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum. Bioinformatics, biochemical analyses, and X-ray crystallography revealed CtaZ as a founding member of a new group of GyrI-like proteins. CtaZ is unique in binding a polythioamide scaffold in a ligand-optimized hydrophobic pocket, thereby confining CTA. By genome mining using CtaZ as a handle, we discovered previously overlooked homologs encoded by diverse members of the phylum Firmicutes, including many pathogens. In addition to characterizing both a new role for a GyrI-like domain in self-resistance and unprecedented thioamide binding, this work aids in uncovering related drug-resistance mechanisms.

An artificial metalloprotein with metal-adaptive coordination sites and Ni-dependent quercetinase activity.

Engineering non-native metal active sites into proteins using canonical amino acids offers many advantages but is hampered by significant challenges. The TIM barrel protein, imidazole glycerol phosphate synthase from the hyperthermophilic organism Thermotoga maritima (tHisF), is well-suited for the construction of artificial metalloenzymes by this approach. To this end, we have generated a tHisF variant (tHisFEHH) with a Glu/His/His motif for metal ion coordination. Crystal structures of ZnII:tHisFEHH and NiII:tHisFEHH reveal that both metal ions bind to the engineered histidines. However, the two metals bind at distinct sites with different geometries, demonstrating the adaptability of tHisF. Only ZnII additionally ligates the Glu residue and adopts a tetrahedral geometry. The pseudo-octahedral NiII site comprises the two His and a native Ser residue. NiII:tHisFEHH catalyzes the oxidative cleavage of the flavanols quercetin and myricetin, providing an unprecedented example of an artificial metalloprotein with quercetinase activity.

Pathogenic bacteria remodel central metabolic enzyme to build a cyclopropanol warhead.

Bacteria of the Burkholderia pseudomallei (BP) group pose a global health threat, causing the infectious diseases melioidosis, a common cause of pneumonia and sepsis, and glanders, a contagious zoonosis. A trait of BP bacteria is a conserved gene cluster coding for the biosynthesis of polyketides (malleicyprols) with a reactive cyclopropanol unit that is critical for virulence. Enzymes building this warhead represent ideal targets for antivirulence strategies but the biochemical basis of cyclopropanol formation is unknown. Here we describe the formation of the malleicyprol warhead. We show that BurG, an unusual NAD+-dependent member of the ketol-acid reductoisomerase family, constructs the strained cyclopropanol ring. Biochemical assays and a suite of eight crystal structures of native and mutated BurG with bound analogues and inhibitors provide snapshots of each step of the complex reaction mechanism, involving a concealed oxidoreduction and a C-S bond cleavage. Our findings illustrate a remarkable case of neofunctionalisation, where a biocatalyst from central metabolism has been evolutionarily repurposed for warhead production in pathogens.

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria.

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes generate natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the XP biosynthetic gene clusters (BGCs) has limited the understanding of how natural products affect interactions between the organisms. Here we combine pangenome and sequence similarity networks to analyse BGCs from 45 XP strains that cover all sequenced strains in our collection and represent almost all XP taxonomy. The identified 1,000 BGCs belong to 176 families. The most conserved families are denoted by 11 BGC classes. We homologously (over)express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, virulence factors against insects, metallophores and insect immunosuppressants. These findings explain the functional basis of bacterial natural products in this tripartite relationship.

Structural insights into cooperative DNA recognition by the CCAAT-binding complex and its bZIP transcription factor HapX.

The heterotrimeric CCAAT-binding complex (CBC) is a fundamental eukaryotic transcription factor recognizing the CCAAT box. In certain fungi, like Aspergilli, the CBC cooperates with the basic leucine zipper HapX to control iron metabolism. HapX functionally depends on the CBC, and the stable interaction of both requires DNA. To study this cooperative effect, X-ray structures of the CBC-HapX-DNA complex were determined. Downstream of the CCAAT box, occupied by the CBC, a HapX dimer binds to the major groove. The leash-like N terminus of the distal HapX subunit contacts the CBC, and via a flexible polyproline type II helix mediates minor groove interactions that stimulate sequence promiscuity. In vitro and in vivo mutagenesis suggest that the structural and functional plasticity of HapX results from local asymmetry and its ability to target major and minor grooves simultaneously. The latter feature may also apply to related transcription factors such as yeast Hap4 and distinct Yap family members.