Showing papers by "Christopher T. Walsh published in 2009"
TL;DR: The emergence of multidrug resistance among the latest generation of pathogens suggests that the discovery of new scaffolds should be a priority, and promising approaches to scaffold discovery are emerging.
Abstract: Antibiotic-resistant strains of pathogenic bacteria are increasingly prevalent in hospitals and the community. New antibiotics are needed to combat these bacterial pathogens, but progress in developing them has been slow. Historically, most antibiotics have come from a small set of molecular scaffolds whose functional lifetimes have been extended by generations of synthetic tailoring. The emergence of multidrug resistance among the latest generation of pathogens suggests that the discovery of new scaffolds should be a priority. Promising approaches to scaffold discovery are emerging; they include mining underexplored microbial niches for natural products, designing screens that avoid rediscovering old scaffolds, and repurposing libraries of synthetic molecules for use as antibiotics.
1,620 citations
TL;DR: It is demonstrated that cyclopamine, a widely used Hh antagonist, induces a cilial translocation of Smo similar to that reported for Shh ligand and several Hh agonists.
Abstract: Smoothened (Smo), a 7-pass transmembrane protein, is essential for transduction of a Hedgehog (Hh) signal across the cell membrane. Smo is also the principle therapeutic target for several candidate drugs in the treatment of Hh-related diseases. Mammalian Smo translocates to the primary cilium in response to Sonic hedgehog (Shh) ligand-mediated signaling. A mechanistic understanding of Smo translocation and its interactions with drug candidates is pivotal to our understanding of Hh signaling and the design, development and application of successful drugs. We established a system in which Smo was dual-labeled with GFP and a 12-aa tag whose recognition by an enzymatic process enables the posttranslational labeling of Smo in the cell membrane within the living cell. These tools enable the simultaneous visualization of all cellular Smo, and more specifically, the cell membrane restricted subpopulation. Using this system, we demonstrate that cyclopamine, a widely used Hh antagonist, induces a cilial translocation of Smo similar to that reported for Shh ligand and several Hh agonists. In contrast, other antagonists abrogate the Shh-induced, cilial translocation of Smo. We present evidence that the majority of cilial-localized Smo originates from an intracellular source and may traffic to the primary cilium through an intraflagellar transport (IFT) pathway.
204 citations
TL;DR: Testing for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D and extended X-ray absorption fine structure spectroscopy calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.
Abstract: Aliphatic halogenases activate O2, cleave α-ketoglutarate (αKG) to CO2 and succinate, and form haloferryl [X−Fe(IV)═O; X = Cl or Br] complexes that cleave aliphatic C−H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related αKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O2 to form the C−H bond-cleaving ferryl complex is “triggered” by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presenc...
199 citations
TL;DR: It is shown that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H-abstraction proficiency for halogen-rebound selectivity, and substrate-intermediate disposition and the carboxylate → halide ligand swap combine to specify theHalogenation outcome.
Abstract: The α-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His2(Asp/Glu)1“facial triad” of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X−) coordinates at the vacated site. Halogenation is thought to result from “rebound” of the halogen radical from the X-Fe(III)-OH intermediate produced by hydrogen (H•) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H•-abstraction proficiency for halogen-rebound selectivity. Upon replacement of l-Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid l-norvaline, decay of the chloroferryl intermediate becomes 130× faster and the reaction outcome switches to primarily hydroxylation of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H• abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation to chlorination. Thus, substrate-intermediate disposition and the carboxylate → halide ligand swap combine to specify the halogenation outcome.
189 citations
TL;DR: A biosynthetic gene cluster for thiocillin is reported and it is established that it, and by extension the whole class, is ribosomally synthesized.
Abstract: The thiazolylpeptides are a family of >50 bactericidal antibiotics that block the initial steps of bacterial protein synthesis. Here, we report a biosynthetic gene cluster for thiocillin and establish that it, and by extension the whole class, is ribosomally synthesized. Remarkably, the C-terminal 14 residues of a 52-residue peptide precursor undergo 13 posttranslational modifications to give rise to thiocillin, making this antibiotic the most heavily posttranslationally-modified peptide known to date.
177 citations
TL;DR: Recent breakthrough achievements in both X-ray and NMR spectroscopic studies are reviewed that illuminate the architecture of NRPS PCP domains, PCP-containing didomain-fragments and of a full termination module (C-A-PCP-TE).
163 citations
TL;DR: The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl CoA, and dimethylallyl pyrophosphate by consecutive action of three enzymes, CPAS, CpaD, and CpaO.
Abstract: The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl-CoA, and dimethylallyl pyrophosphate by consecutive action of three enzymes, CpaS, CpaD, and CpaO CpaS is a hybrid, two module polyketide synthase−nonribosomal peptide synthetase (PKS-NRPS) that makes and releases cyclo-acetoacetyl-l-tryptophan (cAATrp), the tetramic acid that serves as substrate for subsequent prenylation and oxidative cyclization to the five ring CPA scaffold The NRPS module in CpaS has a predicted four-domain organization of condensation, adenylation, thiolation, and reductase* (C-A-T-R*), where R* lacks the critical Ser-Tyr-Lys catalytic triad of the short chain dehydrogenase/reductase (SDR) superfamily By heterologous overproduction in Escherichia coli of the 56 kDa Aspergillus flavus CpaS TR* didomain and the single T and R* domains, we demonstrate that CpaS catalyzes a Dieckmann-ty
129 citations
TL;DR: The conventional notion that peptides are poor candidates for orally available drugs because of protease‐sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide‐based frameworks that are synthesized and utilized by Nature.
Abstract: The conventional notion that peptides are poor candidates for orally available drugs because of protease-sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide-based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best-selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic-resistant bacterial pathogens.
106 citations
TL;DR: Through in vivo manipulation of the gene responsible for production of the thiocillin precursor peptide, 65 novel thiOCillin variants are generated, allowing us to explore structure-activity relationships involved in both precursor peptides maturation and antibiotic activity.
Abstract: The thiocillins are natural-product antibiotics derived from ribosomally encoded peptides that undergo extensive posttranslational modifications to yield the mature trithiazolylpyridine-containing macrocyclic compound. Poor pharmacokinetic properties have prevented the clinical use of these highly potent antibiotics. Through in vivo manipulation of the gene responsible for production of the thiocillin precursor peptide, we have generated 65 novel thiocillin variants, allowing us to explore structure-activity relationships involved in both precursor peptide maturation and antibiotic activity.
85 citations
TL;DR: Comparison of the open conformation of CytC3 with the closed conforming of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe—Cl catalyst: the presence of a hydrogen-bonding network between the chloride and surrounding residues, and the existence of a hydrophobic pocket in which the chloride resides.
Abstract: CytC3, a member of the recently discovered class of nonheme Fe(II) and alpha-ketoglutarate (alphaKG)-dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, gamma,gamma-dichloroaminobutyrate. Unlike the majority of the Fe(II)-alphaKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.
77 citations
TL;DR: It is reported that ScyC, previously annotated as a hypothetical protein, catalyzes cyclization and decarboxylation of 2 to generate ketone 5, and Assembly of the cyclopentyl[b]indole structure in this manner has little precedent in the chemical literature.
Abstract: Previous studies of the biosynthetic enzymes involved in the assembly of scytonemin (1), a cyanobacterial sunscreen, have identified β-ketoacid 2 as an important intermediate that is produced by ThDP-dependent enzyme ScyA. We now report that ScyC, previously annotated as a hypothetical protein, catalyzes cyclization and decarboxylation of 2 to generate ketone 5. Assembly of the cyclopentyl[b]indole structure in this manner has little precedent in the chemical literature. Additional mechanistic experiments have revealed that cyclization likely precedes decarboxylation and that the latter event may provide a driving force for cyclopentane formation.
TL;DR: In this study, two putative nonheme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the beta-position of glutamic acid.
Abstract: The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one α-hydroxy acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally produced natural products. They contain d-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative nonheme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the β-position of glutamic acid. KtzO generates threo-l-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone adenylation domain KtzN, which acti...
TL;DR: The results reveal the inherent flexibility of the pseudomonine synthetase and thus provide insight into the evolution of siderophore biosynthetic gene clusters in bacteria.
Abstract: Siderophores play a vital role in the survival of bacteria, as they facilitate the transport of iron in low-concentration environments. Nature employs a variety of coordinating functional groups in siderophore scaffolds as a way of creating structural diversity. We have successfully shown that the pseudomonine synthetase can produce three distinct siderophore natural products and five siderophore-like compounds. The in vitro enzymatic production of acinetobactin has prompted a revision of the reported structure from an oxazoline to an isoxazolidinone. Our results reveal the inherent flexibility of the pseudomonine synthetase and thus provide insight into the evolution of siderophore biosynthetic gene clusters in bacteria.
TL;DR: Structural and kinetic analyses reveal a novel‐peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic‐active site for catalysing dual adenosine triphosphate‐consuming reactions, providing insight into how a distinctive member of the E1 superfamily carries out two‐step activation for generating the peptidyl‐antibiotic MccC7.
Abstract: The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a 'Trojan horse' antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7.
TL;DR: The overproduction and purification of the six proteins VbsACGOLS in the bacterial host Escherichia coli and the reconstitution of the biosynthesis of vicibactin from primary metabolites are reported.
Abstract: To acquire iron, the N(2)-fixing, symbiotic bacterium Rhizobium sp. produce the cyclic trihydroxamate siderophore vicibactin, containing a 30-membered trilactone scaffold. Herein we report the overproduction and purification of the six proteins VbsACGOLS in the bacterial host Escherichia coli and the reconstitution of the biosynthesis of vicibactin from primary metabolites. The flavoprotein VbsO acts as a pathway-initiating l-ornithine N(5)-hydroxylase, followed by VbsA, which transfers (R)-3-hydroxybutyryl- from the CoA thioester to N(5)-hydroxyornithine to yield N(5)-((R)-3-hydroxybutyryl)-N(5)-hydroxy-l-ornithine. VbsL is a PLP-dependent epimerase acting at C(2) of the 10 atom monomer unit. VbsS, a nonribosomal peptide synthetase free-standing module, then activates N(5)-((R)-3-hydroxybutyryl)-N(5)-hydroxy-d-ornithine as the AMP anhydride on the way to cyclotrimerization to the vicibactin scaffold. The last step, tris-acetylation of the C(2) amino group of desacetyl-d-vicibactin to the mature siderophore vicibactin, is catalyzed distributively by VbsC, using three molecules of acetyl-CoA.
TL;DR: It is demonstrated that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines, and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid.
Abstract: The fungal neurotoxin alpha-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca(2+)-ATPase with a unique pentacyclic indole tetramic acid scaffold, is assembled by a three enzyme pathway CpaS, CpaD, and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form beta-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines, and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with 11 homologous fungal Trp-DMATs revealed five regions of conservation, suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity.
TL;DR: To build the dapdiamide antibiotic scaffolding, amidation must occur on the fumaroyl-DAP scaffold, after DdaG action but before DdaF catalysis, an unusual instance of two ligases acting sequentially in untemplated amide bond formations using attack of substrate carboxylates at P(alpha) (AMP-forming) and then at P (gamma) (ADP-forming] of ATP cosubstrates.
Abstract: The enzymes DdaG and DdaF, encoded in the Pantoea agglomerans dapdiamide antibiotic biosynthetic gene cluster, when expressed in Escherichia coli, form the tandem amide bonds of the dapdiamide scaffold at the expense of ATP cleavage. DdaG uses fumarate, 2,3-diaminopropionate (DAP), and ATP to make fumaroyl-AMP transiently on the way to the N(beta)-fumaroyl-DAP regioisomer. Then DdaF acts as a second ATP-dependent amide ligase, but this enzyme cleaves ATP to ADP and P(i) during amide bond formation. However, DdaF will not accept N(beta)-fumaroyl-DAP; the enzyme requires the fumaroyl moiety to be first converted to the fumaramoyl half-amide in N(beta)-fumaramoyl-DAP. DdaF adds Val, Ile, or Leu to the carboxylate of fumaramoyl-DAP to make dapdiamide A, B, or C, respectively. Thus, to build the dapdiamide antibiotic scaffold, amidation must occur on the fumaroyl-DAP scaffold, after DdaG action but before DdaF catalysis. This is an unusual instance of two ligases acting sequentially in untemplated amide bond formations using attack of substrate carboxylates at P(alpha) (AMP-forming) and then at P(gamma) (ADP-forming) of ATP cosubstrates.
TL;DR: The first in vitro characterization of enzymatic ureido-linkage formation which is catalyzed by an NRPS, SylC is reported, which shows that biosynthesis occurs via N-carboxylation to form an initial N- carboxy-aminoacyl-S-Ppant enzyme intermediate which undergoes intramolecular cyclization followed by condensation with a second amino acid to form the ureado-containing dipeptide product.
Abstract: Syringolins are a class of cyclic tripeptide natural products that are potent and irreversible inhibitors of the eukaryotic proteasome. In addition to being hybrid NRPS/PKS molecules, they also feature an unusual ureido-linkage (red) between two amino acid monomers. Here we report the first in vitro characterization of enzymatic ureido-linkage formation which is catalyzed by an NRPS, SylC. Using 13C- and 18O-labeling studies, we show that biosynthesis occurs via N-carboxylation to form an initial N-carboxy-aminoacyl-S-Ppant enzyme intermediate which undergoes intramolecular cyclization followed by condensation with a second amino acid to form the ureido-containing dipeptide product.
TL;DR: A novel approach to discovering new classes of antibiotics againstmultidrug-resistant Gram-negative bacteria, including strains of Klebsiella, Acinetobacter and Pseudomonas is reported.
Abstract: With the rise in resistance that inevitably follows the clinical deployment of an antibiotic, there is a continual need for new antibiotic discovery, development and approval. Food and Drug Administration approvals for Synercid (quinupristin/dalfopristin) in 1999, Zyvox (linezolid) in 2000, and Cubicin (daptomycin) in 2003 have addressed life-threatening infections from drug-resistant Gram-positive bacteria such as Staphylococcus aureus, Streptococcus pneumomiae, and Enterococcus faecalis. However, because these antibiotics are not active against an emerging class of nosocomial pathogens (multidrug-resistant Gram-negative bacteria, including strains of Klebsiella, Acinetobacter and Pseudomonas) there is renewed focus on developing treatments for infections caused by Gram-negative bacteria. To this end, in this issue of PNAS Miller et al. (1) report a novel approach to discovering new classes of antibiotics.
TL;DR: Research into the development of antibiotics that can prevent diseases such as methicillin-resistant Staphylococcus aureus (MRSA) and the role of horizontal gene transfers by plasmids in the adaptation of bacteria to resist antibiotics is described.
Abstract: This article discusses research into the development of antibiotics that can prevent diseases such as methicillin-resistant Staphylococcus aureus (MRSA). Research into the role of horizontal gene transfers by plasmids in the adaptation of bacteria to resist antibiotics is described. The use of genomic research and biotechnology to develop antibiotics is addressed. INSETS: MINING GENES FOR DRUGS;FIGHTING THE FLU;EXPANDING THE SEARCH;NOVEL WAYS TO BEAT BUGS
TL;DR: In this paper, the authors reviewed recent breakthrough achievements in both X-ray and NMR spectroscopic studies that illuminate the architecture of NRPS PCP domains, PCP-containing didomainfragments and of a full termination module (C-A-PCP-TE).
Abstract: Nonribosomal peptides have a variety of medicinal activities including activity as antibiotics, antitumor drugs, immunosuppressives, and toxins. Their biosynthesis on multimodular assembly lines as a series of covalently tethered thioesters, in turn covalently attached on pantetheinyl arms on carrier protein way stations, reflects similar chemical logic and protein machinery to fatty acid and polyketide biosynthesis. While structural information on excised or isolated catalytic adenylation (A), condensation (C), peptidyl carrier protein (PCP) and thioesterase (TE) domains had been gathered over the past decade, little was known about how the NRPS catalytic and carrier domains interact with each other both within and across elongation or termination modules. This Highlight reviews recent breakthrough achievements in both X-ray and NMR spectroscopic studies that illuminate the architecture of NRPS PCP domains, PCP-containing didomain-fragments and of a full termination module (C-A-PCP-TE).
TL;DR: An experiment that allows for a straightforward assignment of NMR resonances, even in large and/or challenging proteins, is presented and resolves ambiguities occurring in traditional methods which involve time-consuming and cumbersome strip comparisons obtained with series of triple-resonance spectra.
Abstract: We present an experiment that allows for a straightforward assignment of NMR resonances, even in large and/or challenging proteins. A single 3D double-TROSY experiment provides three pairs of sequential correlations between two alpha carbons, two amide protons, and two nitrogen nuclei. Thus, all correlated nuclei can readily be visualized within all dimensions of the resulting spectrum, and chain elongation of sequential amino acids can be effected with this single data set. This resolves ambiguities occurring in traditional methods which involve time-consuming and cumbersome strip comparisons obtained with series of triple-resonance spectra. The experiment makes use of the double TROSY technique to account for signal intensity losses during transfer and evolution periods and was tested on a 500 μM sample of the 33 kDa nonribosomal peptide synthetase protein EntB.
TL;DR: Herein, the biogenesis of the hydrindane ring system within coronafacic acid (CFA) has been investigated and reveals that in addition to the canonical polyketide chain elongation and functionalization encoded by type Ipolyketide synthase (PKSs), cascade reactions can take place during assembly line-like biosynthesis.
Abstract: Herein, the biogenesis of the hydrindane ring system within coronafacic acid (CFA) has been investigated. These studies reveal that in addition to the canonical polyketide chain elongation and functionalization encoded by type I polyketide synthase (PKSs), cascade reactions can take place during assembly line-like biosynthesis. Indeed, upon Cfa7-catalyzed Claisen condensation between enzyme-bound malonate and an N-acetylcysteamine (SNAC) thioester, latent reactivity within the elongated enzyme-bound intermediate is unveiled. This reactivity translates into an intramolecular cyclization, which can proceed in a facile manner as observed by the enzyme-independent cyclization of a linear beta-ketothioester intermediate.
TL;DR: A time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record 13C- and 15N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples to delineate the two spectra which would otherwise overlap with one another when acquired together.
Abstract: We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record 13C- and 15N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins.
Journal Article•
TL;DR: Modern medicine is losing the war against disease-causing bacteria that were once considered vanquished, and new approaches to discovering and creating antibiotics are needed to turn the tide.
Abstract: “Superbug Strikes in City” sounds like a horror movie title, but instead it is a headline printed in the October 26, 2007, edition of the New York Post. Twelve days earlier a 12-year-old Brooklyn boy, Omar Rivera, died after a wound he received on the basketball court became infected with methicillinresistant Staphylococcus aureus (MRSA), a bacterium that has become resistant to one of the most potent drug classes in the current antibiotic arsenal. The prospect of healthy people contracting an untreatable bacterial infection may have seemed remote a decade ago, but it has now become a reality. In 2007 a research team led by Monina Klevens at the Centers for Disease Control and Prevention reported that MRSA causes 19,000 deaths every year in the U.S., which is more than HIV/AIDS causes. The number is especially alarming because almost 20 percent of people who contract MRSA die from it, and an increasing number of its victims are young, healthy people who were infected going about everyday activities. The problem was once limited to hospitals or nursing homes, where many people were already vulnerable because of impaired immunity. Even for those who survive, the price of MRSA is high: a patient who contracts it while hospitalized stays an average 10 days longer and costs an additional $30,000. The total annual expenditure on treating MRSA infections in U.S. hospitals is an astounding $3 billion to $4 billion, and staph is just one of the pathogens that are becoming more and more difficult to subdue. Modern medicine is losing the war against disease-causing bacteria that were once considered vanquished, and new approaches to discovering and creating antibiotics are needed to turn the tide.