Sequence-structure-function characterization of the emerging tetracycline destructase family of antibiotic resistance enzymes.

Tetracycline destructases (TDases) are flavin monooxygenases which can confer resistance to all generations of tetracycline antibiotics. The recent increase in the number and diversity of reported TDase sequences enables a deep investigation of the TDase sequence-structure-function landscape. Here, we evaluate the sequence determinants of TDase function through two complementary approaches: (1) constructing profile hidden Markov models to predict new TDases, and (2) using multiple sequence alignments to identify conserved positions important to protein function. Using the HMM-based approach we screened 50 high-scoring candidate sequences in Escherichia coli, leading to the discovery of 13 new TDases. The X-ray crystal structures of two new enzymes from Legionella species were determined, and the ability of anhydrotetracycline to inhibit their tetracycline-inactivating activity was confirmed. Using the MSA-based approach we identified 31 amino acid positions 100% conserved across all known TDase sequences. The roles of these positions were analyzed by alanine-scanning mutagenesis in two TDases, to study the impact on cell and in vitro activity, structure, and stability. These results expand the diversity of TDase sequences and provide valuable insights into the roles of important residues in TDases, and flavin monooxygenases more broadly.

Siderophore Synthetase DesD Catalyzes N-to-C Condensation in Desferrioxamine Biosynthesis.

Desferrioxamine siderophores are assembled by the nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase enzyme DesD via ATP-dependent iterative condensation of three N1-hydroxy-N1-succinyl-cadaverine (HSC) units. Current knowledge of NIS enzymology and the desferrioxamine biosynthetic pathway does not account for the existence of most known members of this natural product family, which differ in substitution patterns of the N- and C-termini. The directionality of desferrioxamine biosynthetic assembly, N-to-C versus C-to-N, is a longstanding knowledge gap that is limiting further progress in understanding the origins of natural products in this structural family. Here, we establish the directionality of desferrioxamine biosynthesis using a chemoenzymatic approach with stable isotope incorporation and dimeric substrates. We propose a mechanism where DesD catalyzes the N-to-C condensation of HSC units to establish a unifying biosynthetic paradigm for desferrioxamine natural products in Streptomyces.

Structure of anhydrotetracycline-bound Tet(X6) reveals the mechanism for inhibition of type 1 tetracycline destructases.

Inactivation of tetracycline antibiotics by tetracycline destructases (TDases) remains a clinical and agricultural threat. TDases can be classified as type 1 Tet(X)-like TDases and type 2 soil-derived TDases. Type 1 TDases are widely identified in clinical pathogens. A combination therapy of tetracycline and a TDase inhibitor is much needed to rescue the clinical efficacy of tetracyclines. Anhydrotetracycline is a pan-TDase inhibitor that inhibits both type 1 and type 2 TDases. Here, we present structural, biochemical, and phenotypic evidence that anhydrotetracycline binds in a substrate-like orientation and competitively inhibits the type 1 TDase Tet(X6) to rescue tetracycline antibiotic activity as a sacrificial substrate. Anhydrotetracycline interacting residues of Tet(X6) are conserved within type 1 TDases, indicating a conserved binding mode and mechanism of inhibition. This mode of binding and inhibition is distinct from anhydrotetracycline's inhibition of type 2 TDases. This study forms the framework for development of next-generation therapies to counteract enzymatic tetracycline resistance.

Structure-Based Design of Bisubstrate Tetracycline Destructase Inhibitors That Block Flavin Redox Cycling.

Tetracyclines (TCs) are an important class of antibiotics threatened by an emerging new resistance mechanism─enzymatic inactivation. These TC-inactivating enzymes, also known as tetracycline destructases (TDases), inactivate all known TC antibiotics, including drugs of last resort. Combination therapies consisting of a TDase inhibitor and a TC antibiotic represent an attractive strategy for overcoming this type of antibiotic resistance. Here, we report the structure-based design, synthesis, and evaluation of bifunctional TDase inhibitors derived from anhydrotetracycline (aTC). By appending a nicotinamide isostere to the C9 position of the aTC D-ring, we generated bisubstrate TDase inhibitors. The bisubstrate inhibitors have extended interactions with TDases by spanning both the TC and presumed NADPH binding pockets. This simultaneously blocks TC binding and the reduction of FAD by NADPH while "locking" TDases in an unproductive FAD "out" conformation.

In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii.

Siderophores produced via nonribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. Improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central l-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of l-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of l-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of l-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of l-Ser, DHB, and l-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (l-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS "branching" in the presence of a terminating C-T-C motif (FbsG).

An acyl-adenylate mimic reveals the structural basis for substrate recognition by the iterative siderophore synthetase DesD.

Siderophores are conditionally essential metabolites used by microbes for environmental iron sequestration. Most Streptomyces strains produce hydroxamate-based desferrioxamine (DFO) siderophores composed of repeating units of N1-hydroxy-cadaverine (or N1-hydroxy-putrescine) and succinate. The DFO biosynthetic operon, desABCD, is highly conserved in Streptomyces; however, expression of desABCD alone does not account for the vast structural diversity within this natural product class. Here, we report the in vitro reconstitution and biochemical characterization of four DesD orthologs from Streptomyces strains that produce unique DFO siderophores. Under in vitro conditions, all four DesD orthologs displayed similar saturation steady-state kinetics (Vmax = 0.9-2.5 µM⋅min-1) and produced the macrocyclic trimer DFOE as the favored product, suggesting a conserved role for DesD in the biosynthesis of DFO siderophores. We further synthesized a structural mimic of N1-hydroxy-N1-succinyl-cadaverine (HSC)-acyl-adenylate, the HSC-acyl sulfamoyl adenosine analog (HSC-AMS), and obtained crystal structures of DesD in the ATP-bound, AMP/PPi-bound, and HSC-AMS/Pi-bound forms. We found HSC-AMS inhibited DesD orthologs (IC50 values = 48-53 µM) leading to accumulation of linear trimeric DFOG and di-HSC at the expense of macrocyclic DFOE. Addition of exogenous PPi enhanced DesD inhibition by HSC-AMS, presumably via stabilization of the DesD-HSC-AMS complex, similar to the proposed mode of adenylate stabilization where PPi remains buried in the active site. In conclusion, our data suggest that acyl-AMS derivatives may have utility as chemical probes and bisubstrate inhibitors to reveal valuable mechanistic and structural insight for this unique family of adenylating enzymes.

Multifunctional P450 Monooxygenase CftA Diversifies the Clifednamide Pool through Tandem C-H Bond Activations.

Polycyclic tetramate macrolactams (PTMs) are a class of structurally complex hybrid polyketide-nonribosomal peptide (PK-NRP) natural products produced by diverse bacteria. Several PTMs display pharmaceutically interesting bioactivities, and the early stages of PTM biosynthesis involving polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymology are well studied. However, the timing and mechanisms of post PKS-NRPS oxidations by P450 monooxygenases encoded in PTM biosynthetic gene clusters (BGCs) remain poorly characterized. Here we demonstrate that CftA, encoded in clifednamide-type PTM BGCs, is a multifunctional P450 monooxygenase capable of converting the C29-C30 ethyl side chain of ikarugamycin to either a C29-C30 methyl ketone or a C29-C30 hydroxymethyl ketone through C-H bond activation, resulting in the formation of clifednamide A or clifednamide C, respectively. We also report the complete structure of clifednamide C solved via multidimensional NMR (COSY, HSQC, HMBC, NOESY, and TOCSY) using material purified from an engineered Streptomyces strain optimized for production. Finally, the in vitro reconstitution of recombinant CftA catalytic activity revealed the oxidation cascade for sequential conversion of ikarugamycin to clifednamide A and clifednamide C. Our findings confirm prior genetics-based predictions on the origins of clifednamide complexity via P450s encoded in PTM BGCs and place CftA into a growing group of multifunctional P450s that tailor PTM natural products through late-stage regioselective C-H bond activation.

Structural and Biochemical Characterization of the Flavin-Dependent Siderophore-Interacting Protein from Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen with a high mortality rate due to multi-drug-resistant strains. The synthesis and uptake of the iron-chelating siderophores acinetobactin (Acb) and preacinetobactin (pre-Acb) have been shown to be essential for virulence. Here, we report the kinetic and structural characterization of BauF, a flavin-dependent siderophore-interacting protein (SIP) required for the reduction of Fe(III) bound to Acb/pre-Acb and release of Fe(II). Stopped-flow spectrophotometric studies of the reductive half-reaction show that BauF forms a stable neutral flavin semiquinone intermediate. Reduction with NAD(P)H is very slow (k obs, 0.001 s-1) and commensurate with the rate of reduction by photobleaching, suggesting that NAD(P)H are not the physiological partners of BauF. The reduced BauF was oxidized by Acb-Fe (k obs, 0.02 s-1) and oxazole pre-Acb-Fe (ox-pre-Acb-Fe) (k obs, 0.08 s-1), a rigid analogue of pre-Acb, at a rate 3-11 times faster than that with molecular oxygen alone. The structure of FAD-bound BauF was solved at 2.85 Å and was found to share a similarity to Shewanella SIPs. The biochemical and structural data presented here validate the role of BauF in A. baumannii iron assimilation and provide information important for drug design.

Synthetic Mimics of Native Siderophores Disrupt Iron Trafficking in Acinetobacter baumannii.

Many pathogenic bacteria biosynthesize and excrete small molecule metallophores, known as siderophores, that are used to extract ferric iron from host sources to satisfy nutritional need. Native siderophores are often structurally complex multidentate chelators that selectively form high-affinity octahedral ferric iron complexes with defined chirality recognizable by cognate protein receptors displayed on the bacterial cell surface. Simplified achiral analogues can serve as synthetically tractable siderophore mimics with potential utility as chemical probes and therapeutic agents to better understand and treat bacterial infections, respectively. Here, we demonstrate that synthetic spermidine-derived mixed ligand bis-catecholate monohydroxamate siderophores (compounds 1-3) are versatile structural and biomimetic analogues of two native siderophores, acinetobactin and fimsbactin, produced by Acinetobacter baumannii, a multidrug-resistant Gram-negative human pathogen. The metal-free and ferric iron complexes of the synthetic siderophores are growth-promoting agents of A. baumannii, while the Ga(III)-complexes are potent growth inhibitors of A. baumannii with MIC values <1 µM. The synthetic siderophores compete with native siderophores for uptake in A. baumannii and maintain comparable apparent binding affinities for ferric iron (KFe) and the siderophore-binding protein BauB (Kd). Our findings provide new insight to guide the structural fine-tuning of these compounds as siderophore-based therapeutics targeting pathogenic strains of A. baumannii.

Tetracycline-inactivating enzymes from environmental, human commensal, and pathogenic bacteria cause broad-spectrum tetracycline resistance.

Tetracycline resistance by antibiotic inactivation was first identified in commensal organisms but has since been reported in environmental and pathogenic microbes. Here, we identify and characterize an expanded pool of tet(X)-like genes in environmental and human commensal metagenomes via inactivation by antibiotic selection of metagenomic libraries. These genes formed two distinct clades according to habitat of origin, and resistance phenotypes were similarly correlated. Each gene isolated from the human gut encodes resistance to all tetracyclines tested, including eravacycline and omadacycline. We report a biochemical and structural characterization of one enzyme, Tet(X7). Further, we identify Tet(X7) in a clinical Pseudomonas aeruginosa isolate and demonstrate its contribution to tetracycline resistance. Lastly, we show anhydrotetracycline and semi-synthetic analogues inhibit Tet(X7) to prevent enzymatic tetracycline degradation and increase tetracycline efficacy against strains expressing tet(X7). This work improves our understanding of resistance by tetracycline-inactivation and provides the foundation for an inhibition-based strategy for countering resistance.

Emergence of Ferrichelatase Activity in a Siderophore-Binding Protein Supports an Iron Shuttle in Bacteria.

Siderophores are small-molecule high-affinity multidentate chelators selective for ferric iron that are produced by pathogenic microbes to aid in nutrient sequestration and enhance virulence. In Gram-positive bacteria, the currently accepted paradigm in siderophore-mediated iron acquisition is that effluxed metal-free siderophores extract ferric iron from biological sources and the resulting ferric siderophore complex undergoes diffusion-controlled association with a surface-displayed siderophore-binding protein (SBP) followed by ABC permease-mediated translocation across the cell envelope powered by ATP hydrolysis. Here we show that a more efficient paradigm is possible in Gram-positive bacteria where extracellular metal-free siderophores associate directly with apo-SBPs on the cell surface and serve as non-covalent cofactors that enable the holo-SBPs to non-reductively extract ferric iron directly from host metalloproteins with so-called "ferrichelatase" activity. The resulting SBP-bound ferric siderophore complex is ready for import through an associated membrane permease and enzymatic turnover is achieved through cofactor replacement from the readily available pool of extracellular siderophores. This new "iron shuttle" model closes a major knowledge gap in microbial iron acquisition and defines new roles of the siderophore and SBP as cofactor and enzyme, respectively, in addition to the classically accepted roles as a transport substrate and receptor pair. We propose the formal name "siderophore-dependent ferrichelatases" for this new class of catalytic SBPs.

The structural basis of N-acyl-α-amino-ß-lactone formation catalyzed by a nonribosomal peptide synthetase.

Nonribosomal peptide synthetases produce diverse natural products using a multidomain architecture where the growing peptide, attached to an integrated carrier domain, is delivered to neighboring catalytic domains for bond formation and modification. Investigation of these systems can lead to the discovery of new structures, unusual biosynthetic transformations, and to the engineering of catalysts for generating new products. The antimicrobial ß-lactone obafluorin is produced nonribosomally from dihydroxybenzoic acid and a ß-hydroxy amino acid that cyclizes into the ß-lactone during product release. Here we report the structure of the nonribosomal peptide synthetase ObiF1, highlighting the structure of the ß-lactone-producing thioesterase domain and an interaction between the C-terminal MbtH-like domain with an upstream adenylation domain. Biochemical assays examine catalytic promiscuity, provide mechanistic insight, and demonstrate utility for generating obafluorin analogs. These results advance our understanding of the structural cycle of nonribosomal peptide synthetases and provide insights into the production of ß-lactone natural products.

Crossroads of Antibiotic Resistance and Biosynthesis.

The biosynthesis of antibiotics and self-protection mechanisms employed by antibiotic producers are an integral part of the growing antibiotic resistance threat. The origins of clinically relevant antibiotic resistance genes found in human pathogens have been traced to ancient microbial producers of antibiotics in natural environments. Widespread and frequent antibiotic use amplifies environmental pools of antibiotic resistance genes and increases the likelihood for the selection of a resistance event in human pathogens. This perspective will provide an overview of the origins of antibiotic resistance to highlight the crossroads of antibiotic biosynthesis and producer self-protection that result in clinically relevant resistance mechanisms. Some case studies of synergistic antibiotic combinations, adjuvants, and hybrid antibiotics will also be presented to show how native antibiotic producers manage the emergence of antibiotic resistance.

Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes.

The synthesis and biological evaluation of semisynthetic anhydrotetracycline analogues as small molecule inhibitors of tetracycline-inactivating enzymes are reported. Inhibitor potency was found to vary as a function of enzyme (major) and substrate-inhibitor pair (minor), and anhydrotetracycline analogue stability to enzymatic and nonenzymatic degradation in solution contributes to their ability to rescue tetracycline activity in whole cell Escherichia coli expressing tetracycline destructase enzymes. Taken collectively, these results provide the framework for the rational design of next-generation inhibitor libraries en route to a viable and proactive adjuvant approach to combat the enzymatic degradation of tetracycline antibiotics.

Fimsbactin and Acinetobactin Compete for the Periplasmic Siderophore Binding Protein BauB in Pathogenic Acinetobacter baumannii.

Environmental and pathogenic microbes produce siderophores as small iron-binding molecules to scavenge iron from natural environments. It is common for microbes to produce multiple siderophores to gain a competitive edge in mixed microbial environments. Strains of human pathogenic Acinetobacter baumannii produce up to three siderophores: acinetobactin, baumannoferrin, and fimsbactin. Production of acinetobactin and baumannoferrin is highly conserved among clinical isolates while fimsbactin production appears to be less common. Fimsbactin is structurally related to acinetobactin through the presence of catecholate and phenolate oxazoline metal-binding motifs, and both are derived from nonribosomal peptide assembly lines with similar catalytic domain orientations and identities. Here we report on the chemical, biochemical, and microbiological investigation of fimsbactin and acinetobactin alone and in combination. We show that fimsbactin forms a 1:1 complex with iron(III) that is thermodynamically more stable than the 2:1 acinetobactin ferric complex. Alone, both acinetobactin and fimsbactin stimulate A. baumannii growth, but in combination the two siderophores appear to compete and collectively inhibit bacterial growth. We show that fimsbactin directly competes with acinetobactin for binding the periplasmic siderophore-binding protein BauB suggesting a possible biochemical mechanism for the phenomenon where the buildup of apo-siderophores in the periplasm leads to iron starvation. We propose an updated model for siderophore utilization and competition in A. baumannii that frames the molecular, biochemical, and cellular interplay of multiple iron acquisition systems in a multidrug resistant Gram-negative human pathogen.

Crystal Structure of the Siderophore Binding Protein BauB Bound to an Unusual 2:1 Complex Between Acinetobactin and Ferric Iron.

The critical role that iron plays in many biochemical processes has led to an elaborate battle between bacterial pathogens and their hosts to acquire and withhold this critical nutrient. Exploitation of iron nutritional immunity is being increasingly appreciated as a potential antivirulence therapeutic strategy, especially against problematic multidrug resistant Gram-negative pathogens such as Acinetobacter baumannii. To facilitate iron uptake and promote growth, A. baumannii produces a nonribosomally synthesized peptide siderophore called acinetobactin. Acinetobactin is unusual in that it is first biosynthesized in an oxazoline form called preacinetobactin that spontaneously isomerizes to the final isoxazolidinone acinetobactin. Interestingly, both isomers can bind iron and both support growth of A. baumannii. To address how the two isomers chelate their ferric cargo and how the complexes are used by A. baumannii, structural studies were carried out with the ferric acinetobactin complex and its periplasmic siderophore binding protein BauB. Herein, we present the crystal structure of BauB bound to a bis-tridentate (Fe3+L2) siderophore complex. Additionally, we present binding studies that show multiple variants of acinetobactin bind BauB with no apparent change in affinity. These results are consistent with the structural model that depicts few direct polar interactions between BauB and the acinetobactin backbone. This structural and functional characterization of acinetobactin and its requisite binding protein BauB provides insight that could be exploited to target this critical iron acquisition system and provide a novel approach to treat infections caused by this important multidrug resistant pathogen.

Tetracycline-Inactivating Enzymes.

Tetracyclines have been foundational antibacterial agents for more than 70 years. Renewed interest in tetracycline antibiotics is being driven by advancements in tetracycline synthesis and strategic scaffold modifications designed to overcome established clinical resistance mechanisms including efflux and ribosome protection. Emerging new resistance mechanisms, including enzymatic antibiotic inactivation, threaten recent progress on bringing these next-generation tetracyclines to the clinic. Here we review the current state of knowledge on the structure, mechanism, and inhibition of tetracycline-inactivating enzymes.

Immobilized FhuD2 Siderophore-Binding Protein Enables Purification of Salmycin Sideromycins from Streptomyces violaceus DSM 8286.

Siderophores are a structurally diverse class of natural products common to most bacteria and fungi as iron(III)-chelating ligands. Siderophores, including trihydroxamate ferrioxamines, are used clinically to treat iron overload diseases and show promising activity against many other iron-related human diseases. Here, we present a new method for the isolation of ferrioxamine siderophores from complex mixtures using affinity chromatography based on resin-immobilized FhuD2, a siderophore-binding protein (SBP) from Staphylococcus aureus. The SBP-resin enabled purification of charge positive, charge negative, and neutral ferrioxamine siderophores. Treatment of culture supernatants from Streptomyces violaceus DSM 8286 with SBP-resin provided an analytically pure sample of the salmycins, a mixture of structurally complex glycosylated sideromycins (siderophore-antibiotic conjugates) with potent antibacterial activity toward human pathogenic Staphylococcus aureus (minimum inhibitory concentration (MIC) = 7 nM). Siderophore affinity chromatography could enable the rapid discovery of new siderophore and sideromycin natural products from complex mixtures to aid drug discovery and metabolite identification efforts in a broad range of therapeutic areas.

Mechanistic Basis for ATP-Dependent Inhibition of Glutamine Synthetase by Tabtoxinine-ß-lactam.

Tabtoxinine-ß-lactam (TßL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TßL 3-(S)-hydroxy-ß-lactam (3-HßL) warhead. Phosphorylation of TßL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TßL ß-lactam ring remains intact during enzyme inhibition, making TßL mechanistically distinct from traditional ß-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HßL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.