Showing papers by "Christopher T. Walsh published in 2006"
TL;DR: Christopher T. Walsh is the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (BCMP) at Harvard Medical School and has had extensive experience in academic administration, including Chairmanship of the MIT Chemistry Department and the HMS Biological Chemistry & molecular Pharmacology Department.
Abstract: biotics of the penicillin and cephalosporin families, 3,4 as well as the glycopeptides of the vancomycin family 5 (Figure 1a). * To whom correspondence should be addressed: christopher_walsh@ hms.harvard.edu. † Harvard Medical School. ‡ Harvard University. Christopher T. Walsh is the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (BCMP) at Harvard Medical School. He has had extensive experience in academic administration, including Chairmanship of the MIT Chemistry Department (1982−1987) and the HMS Biological Chemistry & Molecular Pharmacology Department (1987−1995) as well as serving as President and CEO of the Dana Farber Cancer Institute (1992−1995). His research has focused on enzymes and enzyme inhibitors, with recent specialization on antibiotics. He and his group have authored over 590 research papers, a text (Enzymatic Reaction Mechanisms), and two books (Antibiotics: Origins, Actions, Resistance and Posttranslational Modification of Proteins: Expanding Nature’s Inventory). He is a member of the National Academy of Sciences, the Institute of Medicine, and the American Philosophical Society.
1,279 citations
TL;DR: This paper presents several newly discovered antibiotics with unique scaffolds and/or novel mechanisms of action, with the potential to form a basis for new antibiotic classes addressing bacterial targets that are currently underexploited.
Abstract: For the past five decades, the need for new antibiotics has been met largely by semisynthetic tailoring of natural product scaffolds discovered in the middle of the 20(th) century. More recently, however, advances in technology have sparked a resurgence in the discovery of natural product antibiotics from bacterial sources. In particular, efforts have refocused on finding new antibiotics from old sources (for example, streptomycetes) and new sources (for example, other actinomycetes, cyanobacteria and uncultured bacteria). This has resulted in several newly discovered antibiotics with unique scaffolds and/or novel mechanisms of action, with the potential to form a basis for new antibiotic classes addressing bacterial targets that are currently underexploited.
569 citations
TL;DR: Halogenated organic molecules can also arise as natural products in which the carbon -halogen bonds are generated enzymatically, and a brief list of some chlorinated, brominated, and iodinated metabolites is noted below.
Abstract: Halogenated organic molecules can also arise as natural products in which the carbon -halogen bonds are generated enzymatically. To date, more than 4500 halogenated natural products have been discovered, 2 though it is likely to be a substantially incomplete inventory. Enzymatic incorporation of halogens during natural product assembly alters physical properties, including electronic and steric effects that can be consequential for determining the affinity and selectivity of interactions with biological targets. Many of the genes encoding such halogen-incorporating enzymes are embedded in specific biosynthetic gene clusters, enabling coordinate regulation to activate these secondary metabolite pathways. Biological halogenation occurs on a diverse array of organic scaffolds, from terpenes to polyketides to nonribosomal peptides. Within these scaffolds, halogen atoms are incorporated on aliphatic carbons, olefinic centers, and a wide variety of aromatic and heterocyclic rings. A recent inventory of halogen-containing natural products indicated 2300 organochlorines, 2100 organobromines, 120 organoiodines, and 30 organofluorines. 2 The larger representation of chlorinated and brominated metabolites probably reflects the abundance of chloride and bromide ions in microenvironments of terrestrial and marine producer organisms. A brief list of some chlorinated, brominated, and iodinated metabolites is noted below. Among the chlorinated natural products of therapeutic interest are vancomycin ( 1), rebeccamycin ( 2), chlortetracycline (3), and chloramphenicol ( 4; Figure 1A).3-6 The natural products1-3 carry chlorine groups on aromatic rings while chloramphenicol has a 2,2-dichloroacetyl group. As shown in Figure 1B, the cyanobacterial metabolite barbamide (5) has a trichloromethyl substituent and syringomycin E ( 6), produced by the phytotoxicPseudomonas syringae , is monochlorinated on the terminal methyl of a threonyl residue. 7,8 In the biocontrol agents pyrrolnitrin ( 7) and pyoluteorin (8; Figure 1C), also produced by Pseudomonas strains, pyrrole rings are monoand dichlorinated, respectively.9,10 Brominated metabolites are typically produced by marine microorganisms, and three such molecules are shown in Figure 2. The bromoterpene snyderol comes in three isomeric forms (9-11; R, â, and γ), reflecting three modes of quenching an intermediate carbocation. 11,12 Convolutamine A (12) has a tribromoanisole ring presumably reflecting three tandem bromination steps during maturation. 13 The terpenoid product laurallene ( 13) has two C-Br bonds, most notably the terminal bromoallene group. 14
456 citations
TL;DR: The focus of this Highlight is primarily on the construction of pyrrole rings during secondary metabolite formation, and on their derivatization and incorporation into natural products.
384 citations
TL;DR: The structure of SyrB2 is reported with both a chloride ion and αKG coordinated to the iron ion at 1.6 Å resolution, revealing a previously unknown coordination of iron, in which the carboxylate ligand of the facial triad is replaced by a chloride ions.
Abstract: Non-haem Fe(ii)/α-ketoglutarate (αKG)-dependent enzymes harness the reducing power of αKG to catalyse oxidative reactions, usually the hydroxylation of unactivated carbons, and are involved in processes such as natural product biosynthesis, the mammalian hypoxic response, and DNA repair1,2. These enzymes couple the decarboxylation of αKG with the formation of a high-energy ferryl-oxo intermediate that acts as a hydrogen-abstracting species2,3,4. All previously structurally characterized mononuclear iron enzymes contain a 2-His, 1-carboxylate motif that coordinates the iron1,2. The two histidines and one carboxylate, known as the ‘facial triad’, form one triangular side of an octahedral iron coordination geometry. A subclass of mononuclear iron enzymes has been shown to catalyse halogenation reactions, rather than the more typical hydroxylation reaction5,6. SyrB2, a member of this subclass, is a non-haem Fe(ii)/αKG-dependent halogenase that catalyses the chlorination of threonine in syringomycin E biosynthesis5. Here we report the structure of SyrB2 with both a chloride ion and αKG coordinated to the iron ion at 1.6 A resolution. This structure reveals a previously unknown coordination of iron, in which the carboxylate ligand of the facial triad is replaced by a chloride ion.
313 citations
TL;DR: Salmochelins as mentioned in this paper is a modified form of enterobactin, known as salmocohelins, which can evade siderocalin and are less hydrophobic than the original siderophore.
Abstract: Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for bacteria growing in animals because of its hydrophobicity and its sequestration by the mammalian protein siderocalin, a component of the innate immune system. However, pathogenic strains of Escherichia coli and Salmonella use enzymes encoded by the iroA gene cluster to tailor enterobactin by glycosylation and linearization. The resulting modified forms of enterobactin, known as salmochelins, can evade siderocalin and are less hydrophobic than enterobactin, restoring this siderophore's iron-scavenging ability in mammals.
300 citations
TL;DR: A protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that includes construction of target protein fusions with PCP or the ybbR tag and Labeling of the PCP- or theYbbR-tagged proteins in cell lysates or on cell surfaces is described.
Abstract: Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the peptidyl carrier protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that includes (i) expression and purification of Sfp, (ii) synthesis of small-molecule probe–CoA conjugates, (iii) construction of target protein fusions with PCP or the ybbR tag, (iv) labeling PCP- or ybbR-tagged target protein fusions in cell lysates and on live cell surfaces and (v) imaging fluorophore-labeled cell surface receptors by fluorescence microscopy. To follow this protocol, we advise that you allow 3 d for the expression and purification of Sfp phosphopantetheinyl transferase, 1 d for the synthesis and purification of the small-molecule probe–CoA conjugates as the substrates of Sfp, 3 d for the cloning of target protein genes as fusions to the PCP or the ybbR tag in the appropriate plasmids and another 3 d for transfecting cell lines with the plasmids and the expression of PCP- or ybbR-tagged proteins. Labeling of the PCP- or the ybbR-tagged proteins in cell lysates or on cell surfaces should require only 15–30 min.
291 citations
TL;DR: This work shows that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp.
Abstract: Numerous bacteria cope with the scarcity of iron in their microenvironment by synthesizing small iron-scavenging molecules known as siderophores. Mammals have evolved countermeasures to block siderophore-mediated iron acquisition as part of their innate immune response. Secreted lipocalin 2 (Lcn2) sequesters the Escherichia coli siderophore enterobactin (Ent), preventing E. coli from acquiring iron and protecting mammals from infection by E. coli. Here, we show that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp., and Klebsiella pneumoniae, allows bacteria to evade sequestration of Ent by Lcn2. We demonstrate that C-glucosylated derivatives of Ent produced by iroA-encoded enzymes do not bind purified Lcn2, and an iroA-harboring strain of E. coli is insensitive to the growth inhibitory effects of Lcn2 in vitro. Furthermore, we show that mice rapidly succumb to infection by an iroA-harboring strain of E. coli but not its wild-type counterpart, and that this increased virulence depends on evasion of host Lcn2. Our findings indicate that the iroA gene cluster allows bacteria to evade this component of the innate immune system, rejuvenating their Ent-mediated iron-acquisition pathway and playing an important role in their virulence.
283 citations
TL;DR: An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.
Abstract: The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the number of proteins predicted by DNA coding capacities owing to directed posttranslational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation, can be classified according to the type of amino acid side chain modified, the category of the modifying enzyme, and the extent of reversibility. Chemical events such as protein splicing, green fluorescent protein maturation, and proteasome autoactivations also represent posttranslational modifications. An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.
201 citations
TL;DR: The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide/polyketide secondary metabolite, and it is concluded that they act to incorporate an acetate-derived beta-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a Delta(2)-isoprenyl- S-car carrier protein.
Abstract: The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide/polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived β-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a Δ2-isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon β-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.
160 citations
TL;DR: The purple chromobacterial pigment violacein arises by enzymatic oxidation and coupling of two molecules of l-tryptophan to give a rearranged pyrrolidone-containing scaffold in the final pigment.
Abstract: The purple chromobacterial pigment violacein arises by enzymatic oxidation and coupling of two molecules of l-tryptophan to give a rearranged pyrrolidone-containing scaffold in the final pigment. We have purified five contiguously encoded proteins VioA−E after expression in Escherichia coli and demonstrate the full 14-electron oxidation pathway to yield the final chromophore. The flavoenzyme VioA and the heme protein VioB work in conjunction to oxidize and dimerize l-tryptophan to a nascent product that can default to the off pathway metabolite chromopyrrolic acid. In the presence of VioE, the intermediate instead undergoes on-pathway [1,2] indole rearrangement to prodeoxyviolacein. The last two enzymes in the pathway are flavin-dependent oxygenases, VioC and VioD, that act sequentially. VioD hydroxylates one indole ring at the 5-position to yield proviolacein, and VioC then acts on the other indole ring at the 2-position to create the oxindole and complete violacein formation.
TL;DR: In vitro reconstitution of leucine halogenation during barbamide biosynthesis has been accomplished and investigation of the substrate specificities of each of the halogenating enzymes revealed their complementary roles in the generation of trichloroleucine.
Abstract: The in vitro reconstitution of leucine halogenation during barbamide biosynthesis has been accomplished. It has been demonstrated that the triple chlorination of the unactivated pro-R methyl group of the peptidyl carrier protein-tethered l-Leu substrate is carried out by the tandem action of two nonheme iron(II)-dependent halogenases, BarB1 and BarB2. Investigation of the substrate specificities of each of the halogenating enzymes revealed their complementary roles in the generation of trichloroleucine.
TL;DR: A new approach for streamlined detection of substrates, intermediates, and products attached to a phosphopantetheinyl arm of the carrier site, and the facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS-PKS projects with benchtop mass spectrometers.
Abstract: With the emergence of drug resistance and the genomic revolution, there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultralarge enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates, and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gas-phase carrier domains, facile elimination occurs in benchtop and Fourier-transform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to <1000 Da ions for structural assignment of intermediates at <0.007 Da mass accuracy without proteolytic digestion. This “top down” approach quickly illuminated diverse acyl intermediates on the carrier domains of the nonribosoma...
TL;DR: The lack of a thioesterase domain in GliP may account both for the slow release of the dipeptide as the cyclic DKP and for the directed fate of intramolecular cyclization to create the DKP scaffold for subsequent elaboration to gliotoxin.
Abstract: The fungal metabolite gliotoxin has a redox-active disulfide bridge spanning carbons 3 and 6 of a diketopiperazine (DKP) scaffold. The proposed DKP synthetase, GliP, from Aspergillus fumigatus Af293, is a three module (A1-T1-C1-A2-T2-C2-T3) 236 kDa protein that can be overproduced in soluble form in Escherichia coli. Once primed on its three thiolation domains with phosphopantetheine prosthetic groups, GliP activates and tethers l-Phe on T1 and l-Ser on T2, before generating the l-Phe-l-Ser-S-T2 dipeptidyl enzyme intermediate. Release of the dipeptide as the cyclic DKP happens slowly both in wild-type GliP and in enzyme forms where C2 and T3 have been mutationally inactivated. The lack of a thioesterase domain in GliP may account both for the slow release and for the directed fate of intramolecular cyclization to create the DKP scaffold for subsequent elaboration to gliotoxin.
TL;DR: This work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner and accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency.
Abstract: Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert l-lysine to l-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts l-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the α-amine, internal cyclization, and subsequent re-reduction of the cyclic Δ1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that th...
TL;DR: The heterologous expression of staP, staC, and rebC in Escherichia coli and the activity of the corresponding enzymes in constructing the two distinct six-ring scaffolds are described.
Abstract: In the biosynthesis of the antitumor indolocarbazoles rebeccamycin and staurosporine by streptomycetes, assembly of the aglycones involves a complex set of oxidative condensations. Overall formation of aglycones K252c and arcyriaflavin A from their biosynthetic precursor chromopyrrolic acid involves four- and eight-electron oxidations, respectively. This process is catalyzed by the remarkable enzyme StaP, with StaC and RebC acting to direct the level of oxidation in the newly formed five-membered ring. An aryl−aryl coupling reaction is integral to this transformation as well as oxidative decarboxylation of the dicarboxypyrrole moiety of chromopyrrolic acid. Herein we describe the heterologous expression of staP, staC, and rebC in Escherichia coli and the activity of the corresponding enzymes in constructing the two distinct six-ring scaffolds. StaP is a cytochrome P450 enzyme, requiring dioxygen, ferredoxin, flavodoxin NADP+-reductase, and NAD(P)H for activity. StaP on its own converts chromopyrrolic acid...
TL;DR: Kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions, consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH(2), chloride ion, and O(2).
Abstract: The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH2 (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH2 and O2 in the halogenase active site was presumed to form the typical FAD(C4a)−OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)−OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)−OH intermediate. The order of addition of FADH2 and O2 was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Format...
TL;DR: Dissection of regions of carrier proteins that are specifically recognized by upstream and downstream catalytic partner proteins is deciphering the logic for multiprotein assembly line construction of these large classes of natural products.
Abstract: Carrier proteins, 80-100 residues in length, serve as information-rich platforms to present growing acyl and peptidyl chains as covalently tethered phosphopantetheinyl-thioester intermediates during the biosynthesis of fatty acid, polyketide, and nonribosomal natural products. Carrier proteins are recognized both in cis and in trans by partner catalytic domains that effect chain-elongating condensations, redox adjustments, other tailoring steps, and finally kinetically controlled disconnection and release of the mature natural product. Dissection of regions of carrier proteins that are specifically recognized by upstream and downstream catalytic partner proteins is deciphering the logic for multiprotein assembly line construction of these large classes of natural products.
TL;DR: Four adjacent open reading frames, cytC1–C4, were cloned from a cytotrienin-producing strain of a Streptomyces sp.
TL;DR: The TROSY technique was implemented for semiconstant time evolutions in both indirect dimensions, which results in remarkable sensitivity and resolution enhancements, and non-uniform sampling in bothirect dimensions combined with Maximum Entropy (MaxEnt) reconstruction enables such dramatic resolution enhancement while maintaining short measuring times.
Abstract: The initial step of protein NMR resonance assignments typically identifies the sequence positions of 1H-15N HSQC cross-peaks. This is usually achieved by tediously comparing strips of multiple triple-resonance experiments. More conveniently, this could be obtained directly with hNcaNH and hNcocaNH-type experiments. However, in large proteins and at very high fields, rapid transverse relaxation severely limits the sensitivity of these experiments, and the limited spectral resolution obtainable in conventionally recorded experiments leaves many assignments ambiguous. We have developed alternative hNcaNH experiments that overcome most of these limitations. The TROSY technique was implemented for semiconstant time evolutions in both indirect dimensions, which results in remarkable sensitivity and resolution enhancements. Non-uniform sampling in both indirect dimensions combined with Maximum Entropy (MaxEnt) reconstruction enables such dramatic resolution enhancement while maintaining short measuring times. Experiments are presented that provide either bidirectional or unidirectional connectivities. The experiments do not involve carbonyl coherences and thus do not suffer from fast chemical shift anisotropy-mediated relaxation otherwise encountered at very high fields. The method was applied to a 300 microM sample of a 37 kDa fragment of the E. coli enterobactin synthetase module EntF, for which high-resolution spectra with an excellent signal-to-noise ratio were obtained within 4 days each.
TL;DR: It is suggested that helix 3 is a major recognition element in EntB-ArCP and the utility of selection-based approaches for studying NRPS biosynthesis is demonstrated.
Abstract: Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis.
TL;DR: Using HPLC and nanospray-Fourier Transform Mass Spectrometry, it is established that all three domains of PigH undergo post-translational modifications and gained insight into the machinery involved in 2,2-dipyrrole biosynthesis.
Abstract: The red streptomycete metabolite prodigiosin has a unique tripyrrolic structure with two of the three pyrrolyl moieties in tandem. Five enzymes, PigA,G,H,I, and J, are involved in dipyrrole (rings A and B) formation. We have heterologously expressed and purified from Escherichia coli these five enzymes. At first, pyrrole ring A is formed on the peptidyl carrier protein PigG by one of two possible ways: (i) by action of the adenylation domain PigI that transforms l-proline into l-prolyl-AMP and by the flavoprotein dehydrogenase PigA responsible for the four-electron oxidation reaction; (ii) by loading with the pyrrolyl-2-carboxyl-(S)-pantetheinyl moiety from synthetic pyrrolyl-CoA using the phosphopantetheinyl transferase Sfp. Subsequently, pyrrole ring B is constructed by PigH after the transfer of ring A to the ketosynthase of PigJ. PigH consists of three domains: two acyl carrier proteins (ACPs) and a seryltransferase (SerT). Using HPLC and nanospray-Fourier Transform Mass Spectrometry (nFTMS), we established that all three domains of PigH undergo post-translational modifications and gained insight into the machinery involved in 2,2-dipyrrole biosynthesis.
TL;DR: This assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown and is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.
Abstract: For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, thi...
TL;DR: It is concluded that G1027A and M1030A are specifically defective in acyl transfer from T to TE, which defines an interaction surface between these two in cis domains in an NRPS module.
TL;DR: The results suggest that mutations in ARFGEF2 may impair targeted transport of FLNA to the cell surface within neural progenitors along the neuroependyma and that disruption of these cells could contribute to PH formation.
Abstract: Periventricular heterotopia (PH) is a malformation of cortical development characterized by nodules of neurons, ectopically located along the lateral ventricles of the brain. Mutations in the vesicle transport ADP-ribosylation factor guanine exchange factor 2 gene (ARFGEF2 )o r the actin-binding Filamin A (FLNA) gene cause PH. Previous studies have shown that FLNA expression is developmentally regulated, with strongest expression observed along the ventricular zone (VZ) and to a lesser degree in postmitotic neurons in the cortex. Here we characterize the expression patterns for ARFGEF2 within the central nervous systems of human and mouse in order to better understand their potential roles in causing PH. ARFGEF2 mRNA was widely expressed in all cortical layers, especially in the neural precursors of the ventricular and subventricular zones (SVZ) during development, with persistent but diminished expression in adulthood. ARFGEF2 encodes for the protein brefeldin-inhibited guanine exchange factor 2 (BIG2). BIG2 protein immunoreactivity was most strongly localized to the neural progenitors along the neuroependymal lining of the VZ during development, with decreased expression in adulthood. Furthermore, overlapping BIG2 and FLNA expression was greatest in these same neuroependymal cells of human embryonic brain and was co-expressed in progenitors by Western blot. Finally, transfection of a dominant-negative construct of ARFGEF2 in SHSY5Y neuroblastoma cells partially blocked FLNA transport from the Golgi apparatus to the cell membrane. These results suggest that mutations in ARFGEF2 may impair targeted transport of FLNA to the cell surface within neural progenitors along the neuroependyma and that disruption of these cells could contribute to PH formation. J. Comp. Neurol. 494:476 – 484, 2006. © 2005 Wiley-Liss, Inc. Indexing terms: ARFGEF2; Filamin A; periventricular heterotopia
TL;DR: It is shown by combinatorial mutagenesis and selection that the aryl carrier protein of EntB (EntB-ArCP) contains localized protein interaction surfaces, and these results suggest that different protein components recognize different faces of EntBs in the enterobactin synthetase, and designing noncognate carrier protein interactions in PKS and NRPS systems should be possible with very few mutations on a particular carrier protein.
Abstract: Carrier proteins are 80- to 100-residue way stations that are central to polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. Because the biosynthetic intermediates for catalytic operations are presented on carrier proteins as covalently attached thioesters (via a 4'-phosphopantetheine prosthetic group), the specific protein-protein interactions between carrier proteins and other NRPS/PKS domains are critical for high-fidelity conversion to the final product. Here we show by combinatorial mutagenesis and selection that the aryl carrier protein of EntB (EntB-ArCP) contains localized protein interaction surfaces. Our strategy involved random mutagenesis of N-terminal regions of EntB-ArCP, then selection for clones that produce enterobactin by plating onto iron-deficient media. We identified several residues that were highly conserved from our selection, two of which (G242 and D244) constitute an interaction surface on EntB-ArCP for the phosphopantetheinyl transferases (PPTases) EntD and Sfp. This PPTase interface is distinct from a previously characterized interface on EntB-ArCP for the downstream elongation module, EntF. These results suggest that different protein components recognize different faces of EntB-ArCP in the enterobactin synthetase and that the majority of EntB-ArCP surface residues are not involved in these interactions. Therefore, designing noncognate carrier protein interactions in PKS and NRPS systems should be possible with very few mutations on a particular carrier protein.
TL;DR: It is suggested that IroB and IroE enhance the ability of Ent-producing pathogens to acquire iron in membrane-rich microenvironments by increasing the rate of glucosylation and macrolactone hydrolysis.
Abstract: Enterobactin (Ent), a prototypic bacterial siderophore, is modified by both the C-glucosyltransferase IroB and the macrolactone hydrolase IroE in pathogenic bacteria that contain the iroA cluster. To investigate the possible effects of glucosylation and macrolactone hydrolysis on the physical properties of Ent, the membrane affinities and iron acquisition rates of Ent and Ent-derived siderophores were measured. The data obtained indicate that Ent has a high membrane affinity (K(x) = 1.5 x 10(4)) similar to that of ferric acinetoferrin, an amphiphile containing two eight-carbon hydrophobic chains. Glucosylation and macrolactone hydrolysis decrease the membrane affinity of Ent by 5-25-fold. Furthermore, in the presence of phospholipid vesicles, the iron acquisition rate is significantly increased by glucosylation and macrolactone hydrolysis, due to the resultant decrease in membrane sequestration of the siderophore. These results suggest that IroB and IroE enhance the ability of Ent-producing pathogens to acquire iron in membrane-rich microenvironments.
TL;DR: The Fe halogenases represent a new branch of the O2 and a-ketoglutarate-decarboxylating superfamily and are powerful enough to halogenate unactivated carbon centers on aminoacyl groups tethered to nonribosomal peptide synthetase assembly lines.
Abstract: Biosynthetic tailoring of nonribosomal peptide and polyketide natural products can enhance their biological activities. Tailoring enzymes can introduce alkyl, acyl, or glycosyl groups onto natural product scaffolds and can oxidize or halogenate biosynthetic intermediates. Chlorinated and brominated molecules make up more than 95% of the more than 4500 known halogenated metabolites. Chloro and bromo substituents are frequently found on aromatic and heteroaromatic rings, and many terpene scaffolds are also brominated and chlorinated by marine microorganisms. Halogenating enzymes discovered to date fall into two categories based on their utilization either of hydrogen peroxide (haloperoxidases) or molecular oxygen (halogenases) as required oxidants. Haloperoxidases can contain either heme iron or a vanadate cofactor, thought to generate enzyme-bound hapohalite equivalents as proximal OCl or OBr. 8] The O2-utilizing halogenases are typically found embedded in biosynthetic gene clusters ; this suggests a tailoring role in specific natural product assembly. This second class of enzymes uses either FADH2 or non-heme Fe II to activate chloride or bromide oxidatively. The flavoproteins work on electron-rich aromatic and heteroaromatic substrates. 12] The Fe halogenases represent a new branch of the O2 and a-ketoglutarate-decarboxylating superfamily and are powerful enough to halogenate unactivated carbon centers on aminoacyl groups tethered to nonribosomal peptide synthetase assembly lines. Thus, the 4-Cl-l-Thr residue in the phytotoxic liACHTUNGTRENNUNGpo ACHTUNGTRENNUNGdepsipeptide syringomycin E (1) is generated by the nonheme Fe halogenase SyrB2 (Scheme 1). Chlorination occurs on the threonyl skeleton only while it is linked via a thioester to a peptidyl carrier protein domain. Remarkably, the cyclopropane ring in the amino acid coronamic acid arises by a similar g-chlorination of an l-allo-Ile-S-protein by the halogenase CmaB. The g-chloride is then displaced intramolecularly by a thioester enolate by action of CmaC. Therefore, the CmaBmediated chlorination is cryptic in cyclopropane formation. In surveying natural products in which biological chlorination is likely to have occurred at an unactivated carbon center, the remarkable functionalization of the two prochiral methyl groups of leucine to yield the regioand stereospecific generation of a trichloromethyl group in the biosynthesis of the cyanobacterial metabolites barbamide (2), dysidenin (3), and dysideathiazole (4 ; Scheme 2A) suggests analogies with the above Fe halogenases. Indeed, the barbamide biosynthetic gene cluster has been sequenced and contains two genes (barB1 and barB2) that encode proteins homologous to SyrB2, but no activity has yet been reported. barB1 and barB2 homologues have also been found in the dysidenin and dysideathiazole producers (dysB1/dysB2 ; one pair in each producer). Only a few natural products contain bromine where biological bromination might have occurred at an unactivated carbon site. One example is lyngbyaloside B (5 ; Scheme 2B), which is also of cyanobacterial origin and could arise from bromination of an unactivated carbon on a biosynthetic precursor. Whether nonheme Fe halogenases are involved in the biosynthesis of brominated natural products is unknown. To evaluate whether the non-heme Fe family of enzymes can indeed carry out bromination and iterative chlorinations at the same carbon site, we have further examined the activity of SyrB2. SyrB2 was shown to act on the threonyl group presented in thioester linkage on the peptidyl carrier protein domain of its partner protein SyrB1. 4-Cl-threonyl-S-SyrB1 was gently hydrolyzed by addition of the thioesterase TycF and detected as the isoindole adduct (Figure 1A). With an almost equimolar ratio of SyrB2/SyrB1, SyrB2 generated a new peak (Figure 1B) that coeluted with the isoindole derivative of authentic 4,4diCl-Thr, synthesized as noted in the Experimental Section. Mass analysis of the new enzymatic product confirmed both the mass and isotope ratios of the diCl-l-Thr isoindole derivative (calcd for [M+H] 392.0 (100%), 394.0 (71%); found 391.7 (100%), 394.1 (68%)). In addition, the ratio of the relative intensity of the peaks corresponding to 4-Cl-l-Thr and 4,4-diCl-lThr for the reaction run in the presence of [Cl] is doubled in the radioactivity detection channel (Figure 1B, trace c) when compared to the UV channel (Figure 1B, trace d) of the same reaction; this is in good agreement with the presence of two chloro substituents. Because the substrate for SyrB2 is a covalent aminoacyl-S protein and the chlorinated product(s) remain covalently tethered, kinetic analysis would be challenging. However, the released monoand diCl-l-Thr products were obtained at a maximum ratio of about 0.38:1 diCl-l-Thr/Cl-l-Thr (Figure 1C). When comparing the ratio of diCl-l-Thr/Cl-l-Thr in Figure 1C and B, it can be seen that the ratio is higher for panel B (1:1 diCl-l-Thr/Cl-l-Thr). This seems to be due to the difference in initial oxygen concentration prior to SyrB2 addition. In panel B, the reaction mixture was anaerobic prior to SyrB2 addition, whereas the buffer was air-saturated for panel C. The slow addition of oxygen after SyrB2 addition increases the product yield, as shown in Figure 1B. This was also observed when demonstrating the oxygen dependency of the reaction in a prior study (Figure 2B in ref. [10]), in which full [a] Dr. F. H. Vaillancourt, Prof. Dr. D. A. Vosburg, Prof. C. T. Walsh Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 (USA) Fax: (+1)617-432-0438 E-mail : christopher_walsh@hms.harvard.edu [b] Dr. F. H. Vaillancourt Present address: Department of Biological Sciences Research and Development, Boehringer Ingelheim (Canada) Ltd Laval, QC, H7S 2G5 (Canada) [c] Prof. Dr. D. A. Vosburg Present address: Department of Chemistry, Harvey Mudd College Claremont, CA 91711 (USA) [] These authors contributed equally to this work.
TL;DR: The crystal structure of IroE from uropathogenic Escherichia coli CFT073 is reported and it is compared to the structure of Fes from Shigella flexneri (PDB entry 2B20), which has similar active sites, but Fes has an additional amino-terminal lid domain.
Abstract: The proliferation of many pathogenic bacteria is limited by the scarcity of soluble iron in their environment. Many of these bacteria scavenge iron by synthesizing and exporting small molecule siderophores that chelate iron. Iron-bound siderophores are subsequently imported for metabolic processing. Three related serine hydrolases have been characterized biochemically in this pathway: Fes, IroD, and IroE. Here, we report the crystal structure of IroE from uropathogenic Escherichia coli CFT073. The native structure and a complex with diisopropyl fluorophosphonate (DFP, a potent serine hydrolase inhibitor) were determined at 2.3 and 1.4 A resolution, respectively. IroE has the typical α/β-hydrolase fold with an atypical catalytic dyad composed of Ser 189 and His 287. Mutation of either residue was detrimental to catalysis. In addition, rather than the typical oxyanion hole composed of backbone amides, IroE employs the atypical guanidinium moiety of Arg 130. Asp 90 anchors Arg 130 in the active site, and mu...
TL;DR: Two time-shared experiments are presented that enable the characterization of all nOes in 1H–13C-ILV methyl-labelled proteins that are otherwise uniformly deuterated and 15N enriched and possibly selectively protonated for distinct residue types.
Abstract: We present two time-shared experiments that enable the characterization of all nOes in 1 H– 13 C-ILV methyl-labelled proteins that are otherwise uniformly deuterated and 15 N enriched and possibly selectively protonated for distinct residue types. A 3D experiment simultaneously provides the spectra of a 3D NOESY-HN-TROSY and of a 3D NOESY-HC-PEP-HSQC. Thus, nOes from any protons to methyl or amide protons are dispersed with respect to 15 N and 13 C chemical shifts, respectively. The single 4D experiment presented here yields simultaneously the four 4D experiments HC-HSQC-NOESYHC-PEP-HSQC, HC-HSQC-NOESY-HN-TROSY, HN-HSQC-NOESY-HN-TROSY and HN-HSQCNOESY-HC-PEP-HSQC. This allows for the unambiguous determination of all nOes involving amide and methyl protons. The method was applied to a ( 1 H, 13 C)-ILV)( 1 H)-FY-(U) 2 H, 15 N) sample of a 37 kDa di-domain of the E. coli enterobactin synthetase module EntF.