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Christopher T Walsh

Publications and source records attributed to Christopher T Walsh.

At least 19 recordsLinked to original sources
PubMed2007

Characterization of the aminocarboxycyclopropane-forming enzyme CmaC.

The biosynthesis of the coronamic acid fragment of the pseudomonal phytotoxin coronatine involves construction of the cyclopropane ring from a gamma-chloro-L-allo-Ile intermediate while covalently tethered as a phosphopantetheinyl thioester to the carrier protein CmaD. The cyclopropane-forming catalyst is CmaC, catalyzing an intramolecular displacement of the gamma-Cl group by the alpha carbon. CmaC can be isolated as a Zn2+ protein with about 10-fold higher activity over the apo form. CmaC will not cyclize free gamma-chloro amino acids or their S-N-acetylcysteamine (NAC) thioester derivatives but will recognize some other carrier protein scaffolds. Turnover numbers of 5 min-1 are observed for Zn-CmaC, acting on gamma-chloro-L-aminobutyryl-S-CmaD, generating 1-aminocyclopropane-1-carbonyl (ACC)-S-CmaD. Products were detected either while still tethered to the phosphopantetheinyl prosthetic arm by mass spectrometry or after thioesterase-mediated release and derivatization of the free amino acid. In D2O, CmaC catalyzed exchange of one deuterium into the aminobutyryl moiety of the gamma-Cl-aminoacyl-S-CmaD, whereas the product ACC-S-CmaD lacked the deuterium, consistent with a competition for a gamma-Cl-aminobutyryl alpha-carbanion between reprotonation and cyclization. CmaC-mediated cyclization yielded solely ACC, resulting from C-C bond formation and no azetidine carboxylate from an alternate N-C cyclization. CmaC could cyclize gamma,gamma-dichloroaminobutyryl to the Cl-ACC product but did not cyclize delta- or epsilon-chloroaminoacyl-S-CmaD substrates.

Amino Acids↗
PubMed2007

Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3.

Enzymatic incorporation of a halogen atom is a common feature in the biosyntheses of more than 4,500 natural products. Halogenation of unactivated carbon centers in the biosyntheses of several compounds of nonribosomal peptide origin is carried out by a class of mononuclear nonheme iron enzymes that require alpha-ketoglutarate (alphaKG, 1), chloride and oxygen. To investigate the ability of these enzymes to functionalize unactivated methyl groups, we characterized the chlorination of the gamma-methyl substituent of L-2-aminobutyric acid (L-Aba, 2) attached to the carrier protein CytC2 by iron halogenase (CytC3) from soil Streptomyces sp. We identified an intermediate state comprising two high-spin Fe(IV) complexes in rapid equilibrium. At least one of the Fe(IV) complexes abstracts hydrogen from the substrate. The demonstration that chlorination proceeds through an Fe(IV) intermediate that cleaves a C-H bond reveals the mechanistic similarity of aliphatic halogenases to the iron- and alphaKG-dependent hydroxylases.

Aminobutyrates↗
PubMed2006

A singular enzymatic megacomplex from Bacillus subtilis.

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

Amino Acid Sequence↗
PubMed2006

GliP, a multimodular nonribosomal peptide synthetase in Aspergillus fumigatus, makes the diketopiperazine scaffold of gliotoxin.

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.

Aspergillus fumigatus↗
PubMed2006

Carrier protein structure and recognition in polyketide and nonribosomal peptide biosynthesis.

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.

Carrier Proteins↗
PubMed2006

In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum.

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.

Bacterial Proteins↗
PubMed2006

Facile detection of acyl and peptidyl intermediates on thiotemplate carrier domains via phosphopantetheinyl elimination reactions during tandem mass spectrometry.

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 nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) found in the biosynthetic pathways of prodigiosin, pyoluteorin, mycosubtilin, nikkomycin, enterobactin, gramicidin, and several proteins from the orphan pksX gene cluster from Bacillus subtilis. By focusing on just those regions undergoing covalent chemistry, the method delivered clean proof for the reversible dehydration of hydroxymethylglutaryl-S-PksL via incorporation of 2H or 18O from the buffer. The facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS-PKS projects with benchtop mass spectrometers.

Acylation↗
PubMed2006

The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2.

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.

Acute-Phase Proteins↗
PubMed2006

Protein assembly line components in prodigiosin biosynthesis: characterization of PigA,G,H,I,J.

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.

Carrier Proteins↗
PubMed2006

Staurosporine and rebeccamycin aglycones are assembled by the oxidative action of StaP, StaC, and RebC on chromopyrrolic acid.

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 into three aglycone products, K252c, arcyriaflavin A, and 7-hydroxy-K252c; in the presence of StaC, K252c is the predominant product, while the presence of RebC directs formation of arcyriaflavin A. (18)O-Labeling studies indicate that the oxygen(s) of the pyrrolinone and maleimide functionalities of the aglycones formed are all derived from dioxygen. This work allowed for the in vitro reconstitution of the full biosynthetic pathway from l-tryptophan to the staurosporine and rebeccamycin aglycones, K252c and 1,11-dichloroarcyriaflavin A.

Carbazoles↗
PubMed2006

Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection.

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.

Escherichia coli Proteins↗
PubMed2006

Characterization of the cereulide NRPS alpha-hydroxy acid specifying modules: activation of alpha-keto acids and chiral reduction on the assembly line.

Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.

Depsipeptides↗
PubMed2006

Bromoenterobactins as potent inhibitors of a pathogen-associated, siderophore-modifying C-glycosyltransferase.

IroB is a C-glycosyltransferase encoded in the iroA cluster. C-Glucosylation of the bacterial siderophore enterobactin by IroB is a strategy some pathogenic bacteria use to evade the host's innate immunity mediated by lipocalin 2 (Lcn2). Without this modification, enterobactin can be tightly bound by host Lcn2, rendering it ineffective as a siderophore. Therefore, IroB inhibitors could be potential antibiotics against iroA-harboring pathogenic bacteria. We used enterobactin analogues to probe the properties of the active site of IroB and found that enterobactin analogues brominated at the C5 positions of the 2,3-dihydroxybenzoyl rings are potent inhibitors of IroB. This finding could lead to the discovery of effective antibiotics targeting iroA-containing bacteria.

Bromine↗
PubMed2006

Installation of the pyrrolyl-2-carboxyl pharmacophore by CouN1 and CouN7 in the late biosynthetic steps of the aminocoumarin antibiotics clorobiocin and coumermycin A1.

The 5-methyl-2-pyrrolylcarbonyl moiety of the aminocoumarin antibiotics clorobiocin and coumermycin A1 is the key pharmacophore for targeting the ATP-binding site of GyrB for inhibition of the bacterial type-II topoisomerase DNA gyrase. During the late stage of clorobiocin and coumermycin A1 biosynthesis, the pyrrolyl-2-carboxyl group is transferred from the peptidyl carrier proteins Clo/CouN1 to the 3'-hydroxyl of the 4-methoxy-L-noviosyl scaffold by the action of the acyltransferases Clo/CouN7. CouN1 and CouN7 have now been heterologously expressed and purified from Escherichia coli. The apo form of CouN1 is converted to the acyl-holo form by loading with pyrrolyl-2-carboxyl-S-pantetheinyl moieties from synthetic pyrrolyl- and 5-methylpyrrolyl-CoAs by the action of the phosphopantetheinyl transferase Sfp. CouN7 acts as an acyltransferase, moving the pyrrole acyl moieties from CouN1 to the noviose sugar of descarbamoylnovobiocin. When the 5-methylpyrrolyl-2-carboxyl-thioester of CouN1 is the cosubstrate, the in vitro product differs from clorobiocin only in a CH3 for Cl group change on the coumarin ring. Double transfer of this acyl moiety by CouN7 to the penultimate intermediate in coumermycin A1 assembly completes that antibiotic biosynthetic pathway.

Acyltransferases↗
PubMed2006

Probing intra- versus interchain kinetic preferences of L-Thr acylation on dimeric VibF with mass spectrometry.

We present a method to probe intra- and interchain activities within dimeric nonribosomal peptide synthetases. Utilizing domain inactivation and analytical mass mutants in conjunction with rapid-quench, mass spectrometry, and a probabilistic kinetic model, we have elucidated the pre-steady-state intra- and interchain rates and the corresponding flux of the acylation of L-Thr onto VibF. Although the intra rate is significantly faster than the inter rate, the data are most consistent with an even flux of covalent substrate loading where neither pathway dominates. These pre-steady-state results confirm previous steady-state in vitro mutant complementation studies of VibF. Extension of this methodology to other dimeric nonribosomal peptide synthetases, and to the related fatty acid and polyketide synthases, will further our biophysical understanding of their acyl-intermediate-processing pathways.

Acylation↗
PubMed2006

Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH.

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 FADH(2) (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 FADH(2) and O(2) 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 FADH(2) and O(2) 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. Formation of flavin intermediates did not require tryptophan, nor were their rates of formation affected by the presence of tryptophan, suggesting that tryptophan likely does not react directly with any flavin intermediates. Furthermore, although final oxidation to FAD occurred with a rate constant of 0.12 s(-)(1), quenched-flow kinetic data showed that the rate constant for 7-chlorotryptophan formation was 0.05 s(-)(1) at 25 degrees C. The kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions. These findings are consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH(2), chloride ion, and O(2).

Actinomycetales↗