Showing papers by "Christopher T. Walsh published in 2008"

The evolution of gene collectives: How natural selection drives chemical innovation

Abstract: DNA sequencing has become central to the study of evolution. Comparing the sequences of individual genes from a variety of organisms has revolutionized our understanding of how single genes evolve, but the challenge of analyzing polygenic phenotypes has complicated efforts to study how genes evolve when they are part of a group that functions collectively. We suggest that biosynthetic gene clusters from microbes are ideal candidates for the evolutionary study of gene collectives; these selfish genetic elements evolve rapidly, they usually comprise a complete pathway, and they have a phenotype—a small molecule—that is easy to identify and assay. Because these elements are transferred horizontally as well as vertically, they also provide an opportunity to study the effects of horizontal transmission on gene evolution. We discuss known examples to begin addressing two fundamental questions about the evolution of biosynthetic gene clusters: How do they propagate by horizontal transfer? How do they change to create new molecules?

The Chemical Versatility of Natural-Product Assembly Lines

Abstract: Microbial natural products of both polyketide and nonribosomal peptide origin have been and continue to be important therapeutic agents as antibiotics, immunosupressants, and antitumor drugs Because the biosynthetic genes for these metabolites are clustered for coordinate regulation, the sequencing of bacterial genomes continues to reveal unanticipated biosynthetic capacity for novel natural products The re-engineering of pathways for such secondary metabolites to make novel molecular variants will be enabled by understanding of the chemical logic and protein machinery in the producer microbes This Account analyzes the chemical principles and molecular logic that allows simple primary metabolite building blocks to be converted to complex architectural scaffolds of polyketides (PK), nonribosomal peptides (NRP), and NRP-PK hybrids The first guiding principle is that PK and NRP chains are assembled as thioseters tethered to phosphopantetheinyl arms of carrier proteins that serve as thiotemplates for chain elongation The second principle is that gate keeper protein domains select distinct monomers to be activated and incorporated with positional specificity into the growing natural product chains Chain growth is via thioclaisen condensations for PK and via amide bond formation for elongating NRP chains Release of the full length acyl/peptidyl chains is mediated by thioesterases, some of which catalyze hydrolysis while others catalyze regiospecific macrocyclization to build in conformational constraints Tailoring of PK and NRP chains, by acylation, alkylation, glycosylation, and oxidoreduction, occurs both during tethered chain growth and after thioesterase-mediated release Analysis of the types of protein domains that carry out chain initiation, elongation, tailoring, and termination steps gives insight into how NRP and PK biosynthetic assembly lines can be redirected to make novel molecules

Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase

Abstract: Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4'-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.

Investigating the initial steps in the biosynthesis of cyanobacterial sunscreen scytonemin.

Abstract: The cyanobacterial natural product scytonemin (1) functions as a sunscreen, absorbing harmful UV-A radiation. Using information from a recently identified gene cluster, we propose a biosynthetic route to this pigment. We also report the characterization of two enzymes, NpR1275 and NpR1276, which are involved in the initial stages of this pathway. A regioselective acyloin reaction between indole-3-pyruvic acid (4) and p-hydroxyphenylpyruvic acid (5) is a key step in assembling the carbon framework of a proposed monomeric scytonemin precursor (2).

Structural basis for the selectivity of the external thioesterase of the surfactin synthetase

Abstract: Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are found in bacteria, fungi, and plants, in the main, producing antibiotics. They are macromolecular machines that rely on the activity of thioesterases to produce biologically active small molecules. They are of particular interest as an assembly system that might be adapted for the production of novel bioactive compounds with possible therapeutic activity. Frueh et al. have solved the structure of a carrier protein — part of the EntF NRPS subunit of enterobactin synthetase — bound to a type I thioesterase. (Type I thioesterases catalyse the final 'release' step of the small molecule from the NRPS or PKS machinery.) The structure reveals that part of the thioesterase can flip open to reveal the carrier-protein binding site of the enzyme; this movement allows the tether of the carrier protein to access the active site. Koglin et al. determined the structure of conformational sub-states of a thioesterase II enzyme. Type II thioesterases are required to regenerate a functional 4'-phosphopantetheine cofactor when it gets mis-primed by reacting with acetyl- and short chain acyl-residues. Comparison with the structures of type I thioesterases reveals the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. The three-dimensional structure of a type II thioesterase, free and in complex with a thiolation domain, is determined. Comparison with type I thioesterases reveal the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds1,2. Type I thioesterases (TEI) catalyse the release step from the assembly line3 of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains4,5,6,7,8,9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems10,11,12. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria13. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes3,14 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.

Maturation of an Escherichia coli Ribosomal Peptide Antibiotic by ATP-Consuming N−P Bond Formation in Microcin C7

Abstract: Synthetic phosphoramidate analogues of nucleosides have been used as enzyme inhibitors for decades and have therapeutic applications in the treatments of HIV and cancer, but little is known about how N-P bonds are fashioned in nature. The heptapeptide MccA undergoes post-translational processing in producer strains of Escherichia coli to afford microcin C7 (MccC7), a "Trojan horse" antibiotic that contains a phosphoramidate linkage to adenosine monophosphate at its C-terminus. We show that the enzyme MccB, encoded by the MccC7 gene cluster, is responsible for formation of the N-P bond in MccC7. This modification requires the consumption of two ATP molecules per MccA peptide and formation and breakdown of a peptidyl-succinimide intermediate.

Tandem Action of the O2- and FADH2-Dependent Halogenases KtzQ and KtzR Produce 6,7-Dichlorotryptophan for Kutzneride Assembly

Abstract: Kutznerides are actinomycete-derived antifungal nonribosomal hexadepsipeptides which are assembled from five unsual nonproteinogenic amino acids and one hydroxy acid. Conserved in all structurally characterized kutznerides is a dichlorinated tricyclic hexahydropyrroloindole postulated to be derived from 6,7-dichlorotryptophan. In this Communication, we identify KtzQ and KtzR as tandem acting FADH2-dependent halogenases that work sequentially on free l-tryptophan to generate 6,7-dichloro-l-tryptophan. Kinetic characterization of these two enzymes has shown that KtzQ (along with the flavin reductase KtzS) acts first to chlorinate at the 7-position of l-tryptophan. KtzR, with a ∼120 fold preference for 7-chloro-l-tryptophan over l-tryptophan, then installs the second chlorine at the 6-position of 7-chloro-l-tryptophan to generate 6,7-dichloro-l-tryptophan. These findings provide further insights into the enzymatic logic of carbon−chloride bond formation during the biosynthesis of halogenated secondary metabo...

beta-Hydroxylation of the aspartyl residue in the phytotoxin syringomycin E: characterization of two candidate hydroxylases AspH and SyrP in Pseudomonas syringae.

Abstract: The pseudomonal phytotoxin syringomycin E and related nonribosomal peptides contain an L- threo-beta-hydroxyaspartyl residue at the eighth position of the lipodepsipeptide backbone as part of a conserved nonproteinogenic tripeptide motif. Informatic analysis of the P. syringae genome suggests only one putative non-heme iron hydroxylase, AspH. On heterologous expression in Escherichia coli AspH shows robust catalytic activity with free L-Asp and L-Asp thioesters to make beta-OH-Asp but yields the erythro diastereomer rather than the threo configuration that is found in syringomycin. Further analysis of the Syr gene cluster indicated that SyrP, previously annotated as the gene regulatory protein for the five-gene Syr cluster, is actually homologous to the known non-heme mononuclear iron hydroxylase TauD. Indeed, purified SyrP acts on Asp tethered as the protein-bound S-pantetheinyl thioester on the eighth module of the SyrE megasynthetase. The hydroxylation gives the anticipated L- threo-3-OH-Asp diastereomer found in syringomycin. The knockout of syrP abolishes the production of the mature syringomycin E, while knockout of aspH has no effect on syringomycin production.

An eight residue fragment of an acyl carrier protein suffices for post-translational introduction of fluorescent pantetheinyl arms in protein modification in vitro and in vivo.

Abstract: Genetically encoded tags for tracking a given protein continue to be of great interest in a multitude of in vitro and in vivo contexts. Acyl carrier proteins, both free-standing and as embedded 80−100 residue domains, contain a specific serine side chain that undergoes post-translational pantetheinylation from CoASH as donor substrate. We have previously used phage display methods to select a 12 residue fragment that retains recognition for modification by the Escherichia coli phosphopantetheinyltransferase (PPTase) AcpS. In this work, we have used 15N-HSQC based NMR titration experiments of a 12-residue peptide substrate with AcpS to identify six specifically interacting residues (S3, L4, D5, M6, W9,and L11) without the formation of any notable secondary structure. Synthesis of a corresponding octapeptide containing 5 of the 6 interacting residues generated a minimal fragment capable of efficient post-translational phosphopantetheinylation. Genetic insertion of this eight residue coding sequence into the...

Biosynthesis of (-)-(1S,2R)-allocoronamic acyl thioester by an FeII-dependent halogenase and a cyclopropane-forming flavoprotein

Abstract: The biosynthetic gene cluster for the kutzneride family of hexapeptidolactones includes the four-gene cassette ktzABCD postulated to generate a nonproteinogenic amino acid. Encoded by this cassette are the nonheme FeII-dependent halogenase KtzD and the acyl-CoA dehydrogenase-like flavoprotein KtzA, proposed to work in conjunction with adenylating protein KtzB and carrier protein KtzC. In the present work, we report the in vitro reconstitution of this four-protein system and identify the final product as (1S,2R)-allocoronamic acid bound in thioester linkage to KtzC. Further analysis of KtzD and KtzA support a biosynthetic pathway that involves KtzD-mediated generation of a γ-chloroisoleucyl intermediate which is cyclized to the final product by KtzA without redox participation of the bound flavin cofactor. This work introduces a new monomer for potential incorporation into nonribosomal peptides and validates the unique strategy for its biosynthesis.

Polyunsaturated fatty-acid-like trans-enoyl reductases utilized in polyketide biosynthesis

Abstract: Polyketide biosynthesis is typically directed by cis-acting catalytic domains. In the case of the Bacillus subtilis secondary metabolite dihydrobacillaene, the cis-acting domains are not sufficient to generate the saturated C14'-C15' bond. In this communication, we identify PksE as a trans-acting enoyl reductase utilized in the biosynthesis of this portion of dihydrobacillaene. PksE is homologous to the enzymes predicted to serve as enoyl reductases in polyunsaturated fatty acid (PUFA) biosynthesis, and we confirmed this functional assignment in vitro. These results suggest a general enoyl reduction pathway in polyketide biosynthesis and a means by which PUFA-like biosynthetic machinery can modulate small-molecule function.

A latent oxazoline electrophile for N-O-C bond formation in pseudomonine biosynthesis.

Abstract: Nitrogen-heteroatom bonds figure prominently in the structural, chemical, and functional diversity of natural products. In the case of Pseudomonas siderophore pseudomonine, an N-O hydroxamate linkage is found uncommonly configured in an isoxazolidinone ring. In an effort to understand the biogenesis of this heterocycle, we have characterized the pseudomonine synthetase in vitro and reconstituted the complete biosynthetic pathway. Our results indicate that the isoxazolidinone of pseudomonine arises from spontaneous rearrangement of an oxazoline precursor. To the best of our knowledge, this is a previously uncharacterized mode of post-assembly line heterocyclization. Our results establish the oxygen of the ubiquitous siderophore hydroxamate functionality as a nucleophile and may be indicative of general strategy for N-O-C bond formation in nature.

Investigations of the MceIJ-catalyzed posttranslational modification of the microcin E492 C-terminus: linkage of ribosomal and nonribosomal peptides to form "trojan horse" antibiotics.

Abstract: MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide syntheta...

Andrimid producers encode an acetyl-CoA carboxyltransferase subunit resistant to the action of the antibiotic

Abstract: Andrimid is a hybrid nonribosomal peptide-polyketide antibiotic that blocks the carboxyl-transfer reaction of bacterial acetyl-CoA carboxylase (ACC) and thereby inhibits fatty acid biosynthesis with submicromolar potency. The andrimid biosynthetic gene cluster from Pantoea agglomerans encodes an admT gene with homology to the acetyl-CoA carboxyltransferase (CT) beta-subunit gene accD. Escherichia coli cells overexpressing admT showed resistance to andrimid. Co-overproduction of AdmT with E. coli CT alpha-subunit AccA allowed for the in vitro reconstitution of an active heterologous tetrameric CT A(2)T(2) complex. A subsequent andrimid-inhibition assay revealed an IC(50) of 500 nM for this hybrid A(2)T(2) in contrast to that of 12 nM for E. coli CT A(2)D(2). These results validated that AdmT is an AccD homolog that confers resistance in the andrimid producer. Mutagenesis studies guided by the x-ray crystal structure of the E. coli A(2)D(2) complex disclosed a single amino acid mutation of AdmT (L203M) responsible for 5-fold andrimid sensitivity (IC(50) = 100 nM). Complementarily, the E. coli AccD mutant M203L became 5-fold more resistant in the CT assays. This observation allowed for bioinformatic identification of several Vibrio cholerae strains in which accD genes encode the Met Leu switches, and their occurrences correlate predictively with sensitivities to andrimid in vivo.

Gatekeeping versus promiscuity in the early stages of the andrimid biosynthetic assembly line.

Abstract: The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal peptide enzyme assembly line in that all thiolation (T) domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-β-Phe-thioester tethered to the AdmI T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from l-Phe to (S)-β-Phe, which in turn is activated by AdmJ and ATP to form (S)-β-Phe-aminoacyl-AMP. AdmJ then transfers the (S)-β-Phe moiety to one of the free-standing T domains, AdmI, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the ...

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

Abstract: The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit α-hydroxyisocaproate (α-HIC). We show here that it arises from initial activation of α-ketoisocaproate (α-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The α-KIC unit is elongated to α-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the α-keto group of α-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the α-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function α- and β-KR acting on disparate positions of a growing chain intermediate.

Morphing peptide backbones into heterocycles.

Abstract: Microbes employ several catalytic strategies to transform conformationally flexible peptide chains into rigidified scaffolds that possess antibiotic or toxin activity. Prominent examples include the biosynthesis of the β-lactam antibiotics of the penicillin and cephalosporin families (1) and the maturation of vancomycin (2) where distinct structural modifications to the nascent peptide chains confer physiological function. In this issue of PNAS, Lee et al. (3) provide the first insight into the chemical structure of streptolysin S (SLS), a hemolytic toxin produced by the human pathogen Streptococcus pyogenes. Its peptide backbone undergoes remarkable posttranslational tailoring, resulting in heterocycle formation and cytolytic activity. Lee et al. further show that a variety of prokaryotes harbor analogous maturation machinery, which suggests widespread use of heterocyclization for altering peptide shape/flexibility and creating functional toxins. This work builds on previous examples where enzymes morph peptide frameworks of both ribosomal and nonribosomal origin.

The FAD cofactor of RebC shifts to an IN conformation upon flavin reduction.

Abstract: RebC is a putative flavin hydroxylase functioning together with RebP to carry out a key step in the biosynthesis of rebeccamycin. To probe the mechanism of flavin-based chemistry in RebC, we solved the structure of RebC with reduced flavin. Upon flavin reduction, the RebC crystal undergoes a change in its unit cell dimension concurrent with a 5 A movement of the isoalloxazine ring, positioning the flavin ring adjacent to the substrate-binding pocket. Additionally, a disordered helix becomes ordered upon flavin reduction, closing off one side of the substrate-binding pocket. This structure, along with previously reported structures, increases our understanding of the RebC enzyme mechanism, indicating that either the reduction of the flavin itself or binding of substrate is sufficient to drive major conformational changes in RebC to generate a closed active site. Our finding that flavin reduction seals the active site such that substrate cannot enter suggests that our reduced flavin RebC structure is off-pa...

Total Biosynthesis: in vitro Reconstitution of Polyketide and Nonribosomal Peptide Pathways

Abstract: This review surveys efforts to reconstitute key steps in polyketide and nonribosomal peptide biosynthetic pathways with purified enzymes and substrates; 344 references are cited.

Non-heme hydroxylase engineering for simple enzymatic synthesis of L-threo-hydroxyaspartic acid.

Abstract: of several proteins in the blood-clotting cascade, and inhibits the function of excitatory amino acid (EAA) transporters as an l-glutamic acid mimic. The last of these functions is of greater importance, as l-glutamate plays a key role as a primary neurotransmitter in the mammalian central nervous system (CNS) and participates in diverse and complex neuronal communication by activating a broad assortment of the EAA receptors. With its potential to overactivate these receptors, l-glutamate can contribute to CNS damage in acute injuries or chronic diseases. Thus, regulation of extracellular l-glutamate concentration, carried out by the EAA transporters, is crucial. A readily available source of 1 could help in further investigations of these transporters and of the complexity of l-glutamate-mediated signaling processes. Various synthetic routes to complex diastereomeric mixtures of erythroand threo-hydroxyaspartic acid have been described previously. These preparations are circuitous and expensive, making a more efficient synthesis of 1 desirable. Enzymatic catalysis provides an alternative approach, but, to the best of our knowledge, no hydroxylase that directly catalyzes the b-hydroxylation of 2 to 1 has been described. We therefore applied a rational protein design approach to fulfill this task. Protein engineering, based on 3D-structural information, has become an accepted tool for the manipulation of enzymes for biocatalysis, and we used this method to alter the substrate specificity of an asparagine oxygenase (AsnO) from l-Asn to lAsp. AsnO, involved in the biosynthesis of calcium-dependent antibiotics (CDAs) in Streptomyces coelicolor, is an Feand a-ketoglutarate-dependent (aKG-dependent) hydroxylase, which exclusively catalyzes the synthesis of l-threo-hydroxyasparagine (3), which is used as a CDA building block. AsnO therefore provides an amino acid with the desired stereochemistry, but does not accept 2 as a substrate. In previous studies, the crystal structure (PDB ID: 2OG7) of AsnO in complexation with 3 and succinate was solved, and the substrate binding residues were identified (Scheme 1). The side chain of resi-

Inhibitors of sterol biosynthesis as Staphylococcus aureus antibiotics.

Abstract: One of the truisms in the medicinal chemistry of antibiotics is that there can never be enough scaffolds and structures, whether of natural or synthetic origin. No matter how successful a new antibiotic is upon clinical introduction, the inevitable selection for resistant microbes will limit its useful lifetime.[1] Resistance to all major classes of antibiotics has been well chronicled, and one of the bacterial pathogens best known for its resistance is Staphylococcus aureus. With high global mortality in human infections, S. aureus has been described as a professional pathogen that has overcome every antibiotic campaign. Methicillin-resistant S. aureus (MRSA) infections are particularly problematic in both community and clinical settings.[2] The aureus designation (Latin for golden) comes from the pigment staphyloxanthin 1,[3] a glycosylated carotenoid whose biosynthetic pathway proceeds through the same early steps as sterol biosynthesis. A recent anti-MRSA drug discovery effort by Liu and coworkers[4] focuses on one step in particular: the head-to-head condensation of two molecules of farnesyl diphosphate (2) to the C30 hydrocarbon dehydrosqualene 3 by the S. aureus enzyme CrtM. This transformation parallels the eukaryotic pathway to cholesterol, including the intermediacy of the cyclopropane-containing molecule presqualene diphosphate 4.[5] However, a distinction occurs in the last step of catalysis: the eukaryotic squalene synthase[6] uses an NADPH-dependent reduction to quench an allylic cation and yield squalene 5 while the staphylococcal enzyme likely abstracts a proton to yield the central olefin in dehydrosqualene. Subsequent enzymatic dehydrogenations create the conjugated chromophore responsible for the golden color in the end product staphyloxanthin.[3] Staphyloxanthin functions as a virulence factor for S. aureus. The conjugated polyene system is thought to quench reactive oxygen species such as superoxide, peroxide, and hypochlorite that are produced by the white cells of vertebrate hosts as they try to kill the staphylococci during the inflammatory response.[7] Thus, while staphyloxanthin biosynthesis does not appear to confer a growth advantage on S. aureus, it increases survival during vertebrate infections and therefore qualifies as a virulence factor. In the search for the next generation of antibiotics, recent efforts have targeted virulence rather than essential gene functions.[8] A team of investigators that includes structural biologists, chemists, and microbiologists have recently discovered that inhibition of the S. aureus dehydrosqualene synthase reduces bacterial survival during infections, offering a proof-of-principle for such a virulence-targeted approach .[4] The effort was based on determining of the x-ray structure of S. aureus CrtM, the dehydrosqualene synthase, after its heterologous expression in E. coli and subsequent purification and crystallization. Since prenyltransferases are involved in terpene and sterol biosynthesis and the posttranslational S-prenylation of proteins, many of these enzymes have been studied in mechanistic detail. The eukaryotic enzyme squalene synthase, a potential drug target for cholesterol-lowering therapy, has been the object of several medicinal chemistry campaigns to identify potent and specific small molecule inhibitors. Based on these efforts, a variety of known inhibitors could be resynthesized and tested as inhibitory ligands for S. aureus CrtM. One of these molecules, a sulfur-containing farnesyl analog 6, could be co-crystallized with CrtM. The x-ray structure of the complex shows two molecules of 6 in the active site, likely defining the orientation of the prenyl side chains of the natural CrtM intermediate presqualene diphosphate.[4] Evaluation of several inhibitors, including other farnesyl diphosphate analogs and amine-containing hydrocarbons that had previously been prepared as mimics of cationic intermediates in the squalene/dehydrosqualene synthase reaction, led to the observation that phosphonosulfonate scaffolds are submicromolar inhibitors of CrtM and could also be co-crystallized. The biarylether phosphonosulfonate 7 was chosen for further evaluation for several reasons: it had a Ki value of 1.5 nM against CrtM, it inhibited staphyloxanthin production when administered to live S. aureus (IC50 = 110 nM), and it had already progressed through preclinical toxiciology and into human clinical studies as a cholesterol-lowering agent without significant adverse effects. Liu and coworkers found that 7 had no effect on the growth of three human cell lines in serum, a cholesterol-rich medium. While 7 caused S. aureus colonies to lose their golden color, it did not inhibit the growth of S. aureus per se, since staphyloxanthin is not essential for growth.[4] The subsequent evaluation of CrtM as a virulence factor involved a model of systemic infection in mice. When 108 colony-forming units of wild-type S. aureus or a crtM knockout were inoculated intraperitoneally (i.p.), the crtM-deficient strain was successfully cleared 72 hours later while the wild-type strain, as expected, was far more resistant to the oxidant-based defenses of the mice. With this result as a backdrop, mice were then treated with 7 for four days and infected i.p. the second day, and bacteria remaining in the kidney were assessed at the end. The treatment worked successfully and the authors conclude that “on average this result corresponds to a 98% decrease in surviving bacteria in the treatment group”.[4] What lessons should be taken from this report? First, the principle that inhibiting a virulence factor in a bacterial pathogen can have a dramatic effect on bacterial survival in a vertebrate host has been proven. (However, since S. aureus is famously difficult to defeat, it will be instructive to see whether repeated passaging of the staphyloxanthin-deficient S. aureus strain leads to compensatory mutations that restore evasion of oxidative host defenses.) A second lesson is that prior medicinal chemistry efforts on mammalian squalene synthases had great utility in this antibiotic drug development program. These efforts have produced a molecular inventory of inhibitors that served as valuable starting points for the evaluation of selectivity for the bacterial enzyme over the host enzyme, ability to penetrate into S. aureus cells, and lack of toxicity in mammalian cells. The definition of a new target is only the beginning of an antibacterial development program, but the existence of compounds that have already been tested in humans lends much confidence to the effort. This story raises the broader question of the utility and advisability of narrow-spectrum vs. broad-spectrum antibiotics. Inhibitors of staphyloxanthin biosynthesis would likely be restricted to treating S. aureus; is this pathogen an important enough target to warrant the development of antibiotics specifically tailored to it? Probably - given the high mortality of S. aureus human infections, three of the antibiotics recently approved by the FDA (quinupristin/dalfopristin, linezolid, and daptomycin) share MRSA as a primary target.[9] In addition, combination therapies may become more prevalent in the face of infections by multidrug-resistant bacteria, so a staphyloxanthin biosynthesis inhibitor might become a useful agent in such an antibacterial cocktail. The recommendations of a U.S. National Research Council committee in 2006 included the development of narrow-spectrum antibiotics to minimize the perturbation of normal microbial flora and to minimize resistance development.[10] While ecologically sound, such a discovery and development strategy will have its own challenges, including real-time diagnostic tests for rapid pathogen identification and a change in mindset about the acceptable market size for a new antibacterial. A breakthrough antibiotic targeted against virulence would advance such a debate.

From thioesters to amides and back: condensation domain reversibility in the biosynthesis of vibriobactin.

Abstract: Vibriobactin synthetase is a four component nonribosomal peptide synthetase (NRPS) system responsible for the biosynthesis of an iron-chelating catechol siderophore that contributes to a system of iron acquisition in Vibrio cholerae—the causative agent of cholera—during vertebrate infections. Vibriobactin synthetase contains two unusual condensation (C) domains responsible for catalyzing amide bond formation between a protein-bound substrate, tethered by thioester linkage to a thiolation (T) domain, and a soluble substrate. VibH, which is a stand alone C domain, catalyzes peptide-bond formation between a terminal amine of norspermidine (NS) and 2,3-dihydroxybenzoate (DHB) loaded on the aryl carrier protein (ArCP) domain of VibB. The second C domain of VibF (C2) then catalyzes sequential amidation of the remaining two amines of NS-DHB with dihydroxyphenyl5-methyloxazaline (DHP-mOx) loaded on the T domain of VibF. VibH and C2 of VibF differ from canonical chain elongation C domains, which catalyze nucleophilic attack of the free amine of a downstream aminoacyl-S-T domain loaded substrate on an upstream nascent peptidyl-S-T domain thioester. Recently, tailoring enzymes involved in the biosynthesis of a variety of biologically active natural products have been shown to be reversible. Glycosyltransferases from the nonribosomal peptide (NRP) vancomycin, the polyketides (PKs) calicheamicin, avermectin, and vicenistatin, and the broad specificity PK glycosyltransferase OleD from the oleandomycin producer Streptomyces antibioticus have been shown to catalyze reversible reactions that allow for sugars and aglycones to be exchanged. The acyltransferase CouN7, which catalyzes the installation of the 5-methylpyrrole pharmacophore in the penultimate step of coumermycin biosynthesis, has also been shown to be reversible, and allows for the transfer of various acyl moieties between aminocoumarin scaffolds. Herein, we demonstrate that like these tailoring enzymes, NRP scaffold generating C domains, specifically VibH and C2 of VibF, also catalyze reversible reactions, and allow for reloading of T domains with acyl moieties and exchange of downstream substrates after amide bond formation. Due to the sequential nature of the two final amidations catalyzed by C2 of VibF, it was of interest to see if the triamide ACHTUNGTRENNUNGvibriobactin could be formed from the diamide intermediate DHP-mOx-NS-DHB. If C2 was a reversible amide-to-thioester catalyst, it would be possible to do an acyl transfer from the amide intermediate to yield free NS-DHB, and DHP-mOx would be reloaded as a thioester onto the T domain of VibF. Then binding of another molecule of DHP-mOx-NS-DHB would allow forward condensation to yield vibriobactin (Scheme 1). Incuba-

Inhibitors of Sterol Biosynthesis as Staphylococcus aureus Antibiotics

Abstract: One of the truisms in the medicinal chemistry of antibiotics is that there can never be enough scaffolds and structures, whether of natural or synthetic origin. No matter how successful a new antibiotic is upon clinical introduction, the inevitable selection for resistant microbes will limit its useful lifetime.[1] Resistance to all major classes of antibiotics has been well chronicled, and one of the bacterial pathogens best known for its resistance is Staphylococcus aureus. With high global mortality in human infections, S. aureus has been described as a professional pathogen that has overcome every antibiotic campaign. Methicillin-resistant S. aureus (MRSA) infections are particularly problematic in both community and clinical settings.[2] The aureus designation (Latin for golden) comes from the pigment staphyloxanthin 1,[3] a glycosylated carotenoid whose biosynthetic pathway proceeds through the same early steps as sterol biosynthesis. A recent anti-MRSA drug discovery effort by Liu and coworkers[4] focuses on one step in particular: the head-to-head condensation of two molecules of farnesyl diphosphate (2) to the C30 hydrocarbon dehydrosqualene 3 by the S. aureus enzyme CrtM. This transformation parallels the eukaryotic pathway to cholesterol, including the intermediacy of the cyclopropane-containing molecule presqualene diphosphate 4.[5] However, a distinction occurs in the last step of catalysis: the eukaryotic squalene synthase[6] uses an NADPH-dependent reduction to quench an allylic cation and yield squalene 5 while the staphylococcal enzyme likely abstracts a proton to yield the central olefin in dehydrosqualene. Subsequent enzymatic dehydrogenations create the conjugated chromophore responsible for the golden color in the end product staphyloxanthin.[3] Staphyloxanthin functions as a virulence factor for S. aureus. The conjugated polyene system is thought to quench reactive oxygen species such as superoxide, peroxide, and hypochlorite that are produced by the white cells of vertebrate hosts as they try to kill the staphylococci during the inflammatory response.[7] Thus, while staphyloxanthin biosynthesis does not appear to confer a growth advantage on S. aureus, it increases survival during vertebrate infections and therefore qualifies as a virulence factor. In the search for the next generation of antibiotics, recent efforts have targeted virulence rather than essential gene functions.[8] A team of investigators that includes structural biologists, chemists, and microbiologists have recently discovered that inhibition of the S. aureus dehydrosqualene synthase reduces bacterial survival during infections, offering a proof-of-principle for such a virulence-targeted approach .[4] The effort was based on determining of the x-ray structure of S. aureus CrtM, the dehydrosqualene synthase, after its heterologous expression in E. coli and subsequent purification and crystallization. Since prenyltransferases are involved in terpene and sterol biosynthesis and the posttranslational S-prenylation of proteins, many of these enzymes have been studied in mechanistic detail. The eukaryotic enzyme squalene synthase, a potential drug target for cholesterol-lowering therapy, has been the object of several medicinal chemistry campaigns to identify potent and specific small molecule inhibitors. Based on these efforts, a variety of known inhibitors could be resynthesized and tested as inhibitory ligands for S. aureus CrtM. One of these molecules, a sulfur-containing farnesyl analog 6, could be co-crystallized with CrtM. The x-ray structure of the complex shows two molecules of 6 in the active site, likely defining the orientation of the prenyl side chains of the natural CrtM intermediate presqualene diphosphate.[4] Evaluation of several inhibitors, including other farnesyl diphosphate analogs and amine-containing hydrocarbons that had previously been prepared as mimics of cationic intermediates in the squalene/dehydrosqualene synthase reaction, led to the observation that phosphonosulfonate scaffolds are submicromolar inhibitors of CrtM and could also be co-crystallized. The biarylether phosphonosulfonate 7 was chosen for further evaluation for several reasons: it had a Ki value of 1.5 nM against CrtM, it inhibited staphyloxanthin production when administered to live S. aureus (IC50 = 110 nM), and it had already progressed through preclinical toxiciology and into human clinical studies as a cholesterol-lowering agent without significant adverse effects. Liu and coworkers found that 7 had no effect on the growth of three human cell lines in serum, a cholesterol-rich medium. While 7 caused S. aureus colonies to lose their golden color, it did not inhibit the growth of S. aureus per se, since staphyloxanthin is not essential for growth.[4] The subsequent evaluation of CrtM as a virulence factor involved a model of systemic infection in mice. When 108 colony-forming units of wild-type S. aureus or a crtM knockout were inoculated intraperitoneally (i.p.), the crtM-deficient strain was successfully cleared 72 hours later while the wild-type strain, as expected, was far more resistant to the oxidant-based defenses of the mice. With this result as a backdrop, mice were then treated with 7 for four days and infected i.p. the second day, and bacteria remaining in the kidney were assessed at the end. The treatment worked successfully and the authors conclude that “on average this result corresponds to a 98% decrease in surviving bacteria in the treatment group”.[4] What lessons should be taken from this report? First, the principle that inhibiting a virulence factor in a bacterial pathogen can have a dramatic effect on bacterial survival in a vertebrate host has been proven. (However, since S. aureus is famously difficult to defeat, it will be instructive to see whether repeated passaging of the staphyloxanthin-deficient S. aureus strain leads to compensatory mutations that restore evasion of oxidative host defenses.) A second lesson is that prior medicinal chemistry efforts on mammalian squalene synthases had great utility in this antibiotic drug development program. These efforts have produced a molecular inventory of inhibitors that served as valuable starting points for the evaluation of selectivity for the bacterial enzyme over the host enzyme, ability to penetrate into S. aureus cells, and lack of toxicity in mammalian cells. The definition of a new target is only the beginning of an antibacterial development program, but the existence of compounds that have already been tested in humans lends much confidence to the effort. This story raises the broader question of the utility and advisability of narrow-spectrum vs. broad-spectrum antibiotics. Inhibitors of staphyloxanthin biosynthesis would likely be restricted to treating S. aureus; is this pathogen an important enough target to warrant the development of antibiotics specifically tailored to it? Probably - given the high mortality of S. aureus human infections, three of the antibiotics recently approved by the FDA (quinupristin/dalfopristin, linezolid, and daptomycin) share MRSA as a primary target.[9] In addition, combination therapies may become more prevalent in the face of infections by multidrug-resistant bacteria, so a staphyloxanthin biosynthesis inhibitor might become a useful agent in such an antibacterial cocktail. The recommendations of a U.S. National Research Council committee in 2006 included the development of narrow-spectrum antibiotics to minimize the perturbation of normal microbial flora and to minimize resistance development.[10] While ecologically sound, such a discovery and development strategy will have its own challenges, including real-time diagnostic tests for rapid pathogen identification and a change in mindset about the acceptable market size for a new antibacterial. A breakthrough antibiotic targeted against virulence would advance such a debate.

The Chemical Versatility of Natural-Product Assembly Lines

Abstract: Microbial natural products of both polyketide and nonribosomal peptide origin have been and continue to be important therapeutic agents as antibiotics, immunosupressants, and antitumor drugs. Because the biosynthetic genes for these metabolites are clustered for coordinate regulation, the sequencing of bacterial genomes continues to reveal unanticipated biosynthetic capacity for novel natural products. The re-engineering of pathways for such secondary metabolites to make novel molecular variants will be enabled by understanding of the chemical logic and protein machinery in the producer microbes. This Account analyzes the chemical principles and molecular logic that allows simple primary metabolite building blocks to be converted to complex architectural scaffolds of polyketides (PK), nonribosomal peptides (NRP), and NRP-PK hybrids. The first guiding principle is that PK and NRP chains are assembled as thioseters tethered to phosphopantetheinyl arms of carrier proteins that serve as thiotemplates for chain elongation. The second principle is that gate keeper protein domains select distinct monomers to be activated and incorporated with positional specificity into the growing natural product chains. Chain growth is via thioclaisen condensations for PK and via amide bond formation for elongating NRP chains. Release of the full length acyl/peptidyl chains is mediated by thioesterases, some of which catalyze hydrolysis while others catalyze regiospecific macrocyclization to build in conformational constraints. Tailoring of PK and NRP chains, by acylation, alkylation, glycosylation, and oxidoreduction, occurs both during tethered chain growth and after thioesterase-mediated release. Analysis of the types of protein domains that carry out chain initiation, elongation, tailoring, and termination steps gives insight into how NRP and PK biosynthetic assembly lines can be redirected to make novel molecules.