Biomedical subjects

Christopher T Walsh

Publications and source records attributed to Christopher T Walsh.

At least 55 records · Page 3Linked to original sources

PubMed2006

How pathogenic bacteria evade mammalian sabotage in the battle for iron.

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.

Animals↗

PubMed2005

Enzymatic generation of the chromopyrrolic acid scaffold of rebeccamycin by the tandem action of RebO and RebD.

During the biosynthesis of the fused six-ring indolocarbazole scaffolds of rebeccamycin and staurosporine, two molecules of L-tryptophan are processed to a pyrrole-containing five-ring intermediate known as chromopyrrolic acid. We report here the heterologous expression of RebO and RebD from the rebeccamycin biosynthetic pathway in Escherichia coli, and tandem action of these two enzymes to construct the dicarboxypyrrole ring of chromopyrrolic acid. Chromopyrrolic acid is oxidized by six electrons compared to the starting pair of L-tryptophan molecules. RebO is an L-tryptophan oxidase flavoprotein and RebD a heme protein dimer with both catalase and chromopyrrolic acid synthase activity. Both enzymes require dioxygen as a cosubstrate. RebD on its own is incompetent with L-tryptophan but will convert the imine of indole-3-pyruvate to chromopyrrolic acid. It displays a substrate preference for two molecules of indole-3-pyruvic acid imine, necessitating a net two-electron oxidation to give chromopyrrolic acid.

Amino Acid Oxidoreductases↗

PubMed2005

Protein posttranslational modifications: the chemistry of proteome diversifications.

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.

Animals↗

PubMed2005

CouO and NovO: C-methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and novobiocin antibiotic biosynthesis.

During the biosynthesis of the streptomycete aminocoumarin antibiotics novobiocin and the dimeric coumermycin A(1), the bicyclic coumarin scaffold is C-methylated adjacent to the phenolic oxygen. The SAM-dependent C-methyltransferases NovO and CouO have been heterologously expressed and purified from Escherichia coli and shown to act after the aminocoumarin ring has been constructed by prior action of Nov/CouHIJK. Neither C-methyltransferase works on the tyrosyl-derived S-pantetheinyl intermediates tethered to NovH or on the subsequently released free aminocoumarin. NovL ligates the aminocoumarin to prenylhydroxybenzoate to yield novobiocic acid, which is the substrate for NovO before it is O-glycosylated by NovM. In coumermycin assembly, the corresponding ligase CouL makes the bis-amide by tandem ligation of two aminocoumarins to a dicarboxypyrrole. CouO works on both the mono- and bis-amides for mono- and di-C-methylation adjacent to the phenolic hydroxyl before it is glycosylated by CouM. Thus, the specific timing of C-methylation in the aminocoumarin antibiotic pathways is established.

Aminocoumarins↗

PubMed2005

Characterization of a new tailoring domain in polyketide biogenesis: the amine transferase domain of MycA in the mycosubtilin gene cluster.

We report the expression and characterization of a truncated form of MycA from the Mycosubtilin gene cluster from Bacillus subtilis. The MycA fragment contains a new amino transferase (AMT) tailoring domain, allowing the first detailed study of a PLP-dependent enzyme operating in cis within the PKS and NRPS biosynthetic paradigm. As the AMT domain acts on covalently bound beta-ketothioesters, and is therefore a single-turnover system, electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS) was used to observe the amine-transfer reaction both for amine donor substrate specificity and to regiospecifically determine enzyme-bound intermediates. We confirm the function of the AMT domain, dissect the mechanistic steps of amine transfer, identify the preferred amine source, and localize the beta-ketothioester substrate during amine transfer.

Bacillus subtilis↗

PubMed2005

Chemoenzymatic approaches for streamlined detection of active site modifications on thiotemplate assembly lines using mass spectrometry.

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

Acylation↗

PubMed2005

In vitro reconstitution of EryCIII activity for the preparation of unnatural macrolides.

EryCIII is a desosaminyltransferase that converts an inactive macrolide precursor to a biologically active antibiotic. It may have potential for the synthesis of unnatural macrolides with useful biological activities. However, it has been difficult to reconstitute the activity of EryCIII in vitro. We report here that purified, inactive EryCIII can be converted to an active catalyst by the addition of another protein encoded in the same gene cluster, EryCII. The EryCII-treated protein retains activity even when EryCII is removed. We also show that AknT, an activator protein from an unrelated gene cluster, is capable of activating EryCIII. Although the mechanism of activation is not yet understood, we have concluded from these experiments that these antibiotic Gtf activator proteins do not function to deliver substrates to EryCIII and do not exert their effects by forming stable complexes with the Gtf during the glycosyltransfer reaction. We report that activated EryCIII is capable of utilizing an alternative sugar donor, so these results lay the groundwork for the production of novel macrolides.

Bacterial Proteins↗

PubMed2005

Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase.

An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl transferase-catalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an alpha-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.

Amino Acid Sequence↗

PubMed2005

Excision of the epothilone synthetase B cyclization domain and demonstration of in trans condensation/cyclodehydration activity.

The epothilones are potent anticancer natural products produced by a polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid involving proteins EpoA-F. The single NRPS module of the epothilone assembly line, EpoB, is a distinct subunit of approximately 160 kDa and consists of four successive domains: cyclization, adenylation, oxidation, and peptidyl carrier protein (Cy-A-Ox-PCP). The cyclization domain is responsible for introduction of the thiazoline heterocycle into the growing polyketide/nonribosomal peptide chain from the precursors malonyl-CoA and cysteine through the multiple steps of condensation, cyclization, and dehydration. This enzyme-bound thiazoline intermediate is subsequently oxidized to a thiazole by the EpoB Ox domain. The EpoB module was dissected to provide 57 kDa EpoB(Cy) and 102 kDa EpoB(A-Ox-PCP) as subunit fragments to evaluate Cy as a free-standing domain. EpoB was reconstituted by these fragments in trans to generate the methylthiazole product. Using this system, apparent kinetic constants for the upstream acyl donor EpoA(ACP) and EpoB(Cy) were determined, providing a measure of affinity for the naturally occurring interface of the amino terminus of EpoB and the EpoA carboxy terminus. Site-directed mutants in excised EpoB(Cy) were prepared and used to examine residues involved in condensation and heterocycle formation. This work demonstrates the ability to define a functional Cy domain by excision from its native NRPS module, and examine both its protein-protein interactions and mechanism of activity.

Amino Acid Sequence↗

PubMed2005

Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis.

The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PltL. Surprisingly, the FADH2-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PltL intermediate and then the 4,5-dichloropyrrolyl-S-PltL product. PltA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FADH2. Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PltL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCl intermediate for capture by the electron-rich C4 and C5 of the heteroaromatic pyrrole moiety.

Amino Acid Sequence↗

PubMed2005

Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis.

Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products. Halogenation can have significant consequences for the bioactivity of these products so there is great interest in understanding the biological catalysts that perform these reactions. Enzymes that halogenate unactivated aliphatic groups have not previously been characterized. Here we report the activity of five proteins-CmaA, CmaB, CmaC, CmaD and CmaE-in the construction of coronamic acid (CMA; 1-amino-1-carboxy-2-ethylcyclopropane), a constituent of the phytotoxin coronatine synthesized by the phytopathogenic bacterium Pseudomonas syringae. CMA derives from l-allo-isoleucine, which is covalently attached to CmaD through the actions of CmaA, a non-ribosomal peptide synthetase module, and CmaE, an unusual acyltransferase. We show that CmaB, a member of the non-haem Fe(2+), alpha-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the gamma-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the gamma-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute gamma-halogenation followed by intramolecular gamma-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation.

Acyltransferases↗

PubMed2005

In vitro characterization of salmochelin and enterobactin trilactone hydrolases IroD, IroE, and Fes.

The iroA locus encodes five genes (iroB, iroC, iroD, iroE, iroN) that are found in pathogenic Salmonella and Escherichia coli strains. We recently reported that IroB is an enterobactin (Ent) C-glucosyltransferase, converting the siderophore into mono-, di-, and triglucosyl enterobactins (MGE, DGE, and TGE, respectively). Here, we report the characterization of IroD and IroE as esterases for the apo and Fe(3+)-bound forms of Ent, MGE, DGE, and TGE, and we compare their activities with those of Fes, the previously characterized enterobactin esterase. IroD hydrolyzes both apo and Fe(3+)-bound siderophores distributively to generate DHB-Ser and/or Glc-DHB-Ser, with higher catalytic efficiencies (k(cat)/K(m)) on Fe(3+)-bound forms, suggesting that IroD is the ferric MGE/DGE esterase responsible for cytoplasmic iron release. Similarly, Fes hydrolyzes ferric Ent more efficiently than apo Ent, confirming Fes is the ferric Ent esterase responsible for Fe(3+) release from ferric Ent. Although each enzyme exhibits lower k(cat)'s processing ferric siderophores, dramatic decreases in K(m)'s for ferric siderophores result in increased catalytic efficiencies. The inability of Fes to efficiently hydrolyze ferric MGE, ferric DGE, or ferric TGE explains the requirement for IroD in the iroA cluster. IroE, in contrast, prefers apo siderophores as substrates and tends to hydrolyze the trilactone just once to produce linearized trimers. These data and the periplasmic location of IroE suggest that it hydrolyzes apo enterobactins while they are being exported. IroD hydrolyzes apo MGE (and DGE) regioselectively to give a single linear trimer product and a single linear dimer product as determined by NMR.

Catalysis↗

PubMed2005

A systematic investigation of the synthetic utility of glycopeptide glycosyltransferases.

Glycosyltransferases involved in the biosynthesis of bacterial secondary metabolites may be useful for the generation of sugar-modified analogues of bioactive natural products. Some glycosyltransferases have relaxed substrate specificity, and it has been assumed that promiscuity is a feature of the class. As part of a program to explore the synthetic utility of these enzymes, we have analyzed the substrate selectivity of glycosyltransferases that attach similar 2-deoxy-L-sugars to glycopeptide aglycons of the vancomycin-type, using purified enzymes and chemically synthesized TDP beta-2-deoxy-L-sugar analogues. We show that while some of these glycopeptide glycosyltransferases are promiscuous, others tolerate only minor modifications in the substrates they will handle. For example, the glycosyltransferases GtfC and GtfD, which transfer 4-epi-L-vancosamine and L-vancosamine to C-2 of the glucose unit of vancomycin pseudoaglycon and chloroorienticin B, respectively, show moderately relaxed donor substrate specificities for the glycosylation of their natural aglycons. In contrast, GtfA, a transferase attaching 4-epi-L-vancosamine to a benzylic position, only utilizes donors that are closely related to its natural TDP sugar substrate. Our data also show that the spectrum of donors utilized by a given enzyme can depend on whether the natural acceptor or an analogue is used, and that GtfD is the most versatile enzyme for the synthesis of vancomycin analogues.

Anti-Bacterial Agents↗

PubMed2005

SyrB2 in syringomycin E biosynthesis is a nonheme FeII alpha-ketoglutarate- and O2-dependent halogenase.

The nine-residue lipodepsipeptide syringomycin E, elaborated as a phytotoxin by Pseudomonas syringae pv. syringae B301D contains a 4-Cl-L-Thr-9 moiety where failure to chlorinate results in a 3-fold drop in biological activity. The proteins SyrB1 and SyrB2 encoded by the biosynthetic cluster are shown to act as a substrate and enzyme pair for SyrB2-mediated chlorination of the aminoacyl-S-enzyme L-Thr-S-SyrB1. SyrB2 is a member of the nonheme Fe(II) alpha-ketoglutarate-dependent enzyme superfamily, and requires O2 and alpha-ketoglutarate as well as chloride ion to carry out monochlorination of the -CH3 group of L-Thr-S-SyrB1. Chlorination of L-Thr-S-SyrB1 was validated by thioesterase-mediated release of L-Thr and 4-Cl-L-Thr, N-derivatization as fluorescent isoindoles, and HPLC separation compared with authentic standards. Incubations with L-[14C]Thr and [36Cl-] as well as MS of the released products further validated identification. Enzymatic oxidative halogenation is a previously uncharacterized reaction type for nonheme Fe(II) enzymes and may be the general mode for biosynthetic halogenation of aliphatic carbons of natural products.

Chlorides↗

PubMed2005

Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme.

Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.

Amino Acid Sequence↗

PubMed2005

Characterization of the aminocoumarin ligase SimL from the simocyclinone pathway and tandem incubation with NovM,P,N from the novobiocin pathway.

Simocyclinone D(8) consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A(1), the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity.

Amide Synthases↗

PubMed2005

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis.

The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a k(cat) of 1.4 min(-1) was observed for the RebF/RebH system but increased at least 10-fold in low-pO2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.

Carbazoles↗

PubMed2005

Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis.

The aminocoumarin antibiotics clorobiocin and coumermycin A(1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. l-Proline is selected and activated as l-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CloN5 and CouN5. Enzymatic oxidation of the prolyl-S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product.

Amino Acid Sequence↗