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C T Walsh

Publications and source records attributed to C T Walsh.

At least 37 records · Page 2Linked to original sources
PubMed2001

Yersiniabactin synthetase: probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HWMP2 subunit.

The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.

Acyl Carrier Protein↗
PubMed2001

Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: novel glycopeptides.

The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltransferases, GtfB, C, D, E, were subcloned from Amycolatopsis orientalis strains that produce chloroeremomycin (GtfB, C) or vancomycin (GtfD, E) into Escherichia coli. After expression and purification, each glycosyltransferase (Gtf) was characterized for activity either with the aglycones (GtfB, E) or the glucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB efficiently glucosylates vancomycin aglycone using UDP-glucose as the glycosyl donor to form desvancosaminyl-vancomycin (vancomycin pseudoaglycone), with k(cat) of 17 min(-1), but has very low glucosylation activity, < or = 0.3 min(-1), for an alternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating both its natural substrate, vancomycin aglycone (k(cat) = 60 min(-1)), and an unnatural substrate, teicoplanin aglycone (k(cat) = 20 min(-1)). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis of UDP-beta-L-4-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both GtfC and GtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD) or the glucosylated teicoplanin scaffold, 7 (GtfD). The GtfC product was the 4-epi-vancosaminyl form of vancomycin. Remarkably, GtfD was able to utilize both an unnatural acceptor, 7, and an unnatural nucleotide sugar donor, UDP-4-epi-vancosamine, to synthesize a novel hybrid teicoplanin/vancomycin glycopeptide. These results establish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate how they can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class of glycopeptide antibiotics.

Actinomycetales↗
PubMed2001

The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition.

Microcin B17 is a 3.1-kDa bactericidal peptide; the putative target of this antibiotic is DNA gyrase. Microcin B17 has no detectable effect on gyrase-catalysed DNA supercoiling or relaxation activities in vitro and is unable to stabilise DNA cleavage in the absence of nucleotides. However, in the presence of ATP, or the non-hydrolysable analogue 5'-adenylyl beta,gamma-imidodiphosphate, microcin B17 stabilises a gyrase-dependent DNA cleavage complex in a manner reminiscent of quinolones, Ca(2+), or the bacterial toxin CcdB. The pattern of DNA cleavage produced by gyrase in the presence of microcin B17 is different from that produced by quinolones and more closely resembles Ca(2+)-mediated cleavage. Several gyrase mutants, including well-known quinolone-resistant mutants, are cross resistant to microcin-induced DNA cleavage. We suggest that microcin exerts its effects through a mechanism that has similarities to those of both the bacterial toxin CcdB and the quinolone antibacterial agents.

Adenosine Triphosphate↗
PubMed2001

Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase.

The 207-kDa polyketide synthase (PKS) module (residues 1-1895) and the 143-kDa nonribosomal peptidyl synthetase (NRPS) module (1896-3163) of the 350-kDa HMWP1 subunit of yersiniabactin synthetase have been expressed in and purified from Escherichia coli in soluble forms to characterize the acyl carrier protein (ACP) domain of the PKS module and the homologous peptidyl carrier protein (PCP(3)) domain of the NRPS module. The apo-ACP and PCP domains could be selectively posttranslationally primed by the E. coli ACPS and EntD phosphopantetheinyl transferases (PPTases), respectively, whereas the Bacillus subtilis PPTase Sfp primed both carrier protein domains in vitro or during in vivo coexpression. The holo-NRPS module but not the holo-PKS module was then selectively aminoacylated with cysteine by the adenylation domain embedded in the HMWP2 subunit of yersiniabactin synthetase, acting in trans. When the acyltransferase (AT) domain of HMWP1 was analyzed for its ability to malonylate the holo carrier protein domains, in cis acylation was first detected. Then, in trans malonylation of the excised holo-ACP or holo-PCP(3)-TE fragments by HMWP1 showed both were malonylated with a 3:1 catalytic efficiency ratio, showing a promiscuity to the AT domain.

Acyl Carrier Protein↗
PubMed2001

Toward efficient analysis of >70 kDa proteins with 100% sequence coverage.

For complete characterization of larger proteins, primary structural analysis by mass spectrometry must be made more efficient. A straightforward approach is illustrated here using two proteins of 159 and 199 kDa with five and nine Lys residues, respectively. These proteins were degraded by Lys-C to mixtures of peptides ranging in size from 5 to 48 kDa, whose multiply charged ions (from electrospray ionization) are far more amenable than the intact proteins to direct interrogation in a Fourier-transform mass spectrometer. For the 199 kDa PchF of approximately 60% purity, an unfractionated Lys-C digest gave 106 isotopic distributions from 71 components (most of which were below 6 kDa); 15% sequence coverage was obtained. For the > 90% pure PchE (159 kDa), complete sequence coverage was obtained from six Lys-C peptides of 5, 8, 26, 32, 40 and 48 kDa, with all but the largest of these measured at isotopic resolution on a 4.7 Tesla instrument. Practical strategies for implementing this characterization strategy on a proteomic scale are considered.

Amino Acid Sequence↗
PubMed2001

Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI.

BACKGROUND: Coumarin group antibiotics, such as novobiocin, coumermycin A1 and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin moiety as their structural core. Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a beta-hydroxy-tyrosine (beta-OH-Tyr) intermediate. Recently the novobiocin gene cluster from Streptomyces spheroides was cloned and sequenced and allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins. RESULTS: Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S. spheroides have been overexpressed in Escherichia coli, purified and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation. The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the holo PCP domain. The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl-S-NovH acyl enzyme. The product beta-OH-tyrosyl-S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive [3H]beta-OH-Tyr and by mass spectrometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of beta-OH-Tyr. The amino acid released was (3R,2S)-3-OH-Tyr by comparison with authentic standards. CONCLUSIONS: This work establishes that NovH and NovI are responsible for the formation of a beta-OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis. Comparable A-PCP/P450 pairs for amino acid beta-hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A1 cluster for tyrosine, and NikP1/NikQ in the nikkomycin cluster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondary metabolites.

Acylation↗
PubMed2001

Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit.

BACKGROUND: Polyketides (PKs) and non-ribosomal peptides (NRPs) are therapeutically important natural products biosynthesized by multimodular protein assembly lines, termed the PK synthases (PKSs) and NRP synthetases (NRPSs), via a similar thiotemplate-mediated mechanism. The potential for productive interaction between these two parallel enzymatic systems has recently been demonstrated, with the discovery that PK/NRP hybrid natural products can be of great therapeutic importance. One newly discovered PK/NRP product, epothilone D from Sorangium cellulosum, has shown great potential as an anti-tumor agent. RESULTS: The chain-initiating methylthiazole ring of epothilone has been generated in vitro as an acyl-S-enzyme intermediate, using five domains from two modules of the polymodular epothilone synthetase. The acyl carrier protein (ACP) domain, excised from the EpoA gene, was expressed in Escherichia coli, purified as an apo protein, and then post-translationally primed with acetyl-CoA using the phosphopantetheinyl transferase enzyme Sfp. The four-domain 150-kDa EpoB subunit (cyclization-adenylation-oxidase-peptidyl carrier protein domains: Cy-A-Ox-PCP) was also expressed and purified in soluble form from E. coli. Post-translational modification with Sfp and CoASH introduced the HS-pantP prosthetic group to the apo-PCP, enabling subsequent loading with L-cysteine to generate the Cys-S-PCP acyl enzyme intermediate. When acetyl-S-ACP (EpoA) and cysteinyl-S-EpoB were mixed, the Cy domain of EpoB catalyzed acetyl transfer from EpoA to the amino group of the Cys-S-EpoB, generating a transient N-Ac-Cys-S-EpoB intermediate that is cyclized and dehydrated to the five-membered ring methylthiazolinyl-S-EpoB. Finally, the FMN-containing Ox domain of EpoB oxidized the dihydro heterocyclic thiazolinyl ring to the heteroaromatic oxidation state, the methylthiazolylcarboxy-S-EpoB. When other acyl-CoAs were substituted for acetyl-CoA in the Sfp-based priming of the apo-CP domain, additional alkylthiazolylcarboxy-S-EpoB acyl enzymes were produced. CONCLUSIONS: These experiments establish chain transfer across a PKS and NRPS interface. Transfer of the acetyl group from the ACP domain of EpoA to EpoB reconstitutes the start of the epothilone synthetase assembly line, and installs and converts a cysteine group into a methyl-substituted heterocycle during this natural product chain growth.

Acyl Coenzyme A↗
PubMed2001

Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines.

Nonribosomal peptide synthetases are large enzyme complexes that synthesize a variety of peptide natural products through a thiotemplated mechanism. Assembly of the peptides proceeds through amino acid loading, amide-bond formation and chain translocation, and finally thioester lysis to release the product. The final products are often heavily modified, however, through methylation, epimerization, hydroxylation, heterocyclization, oxidative cross-linking and attachment of sugars. These activities are the province of specialized enzymes (either embedded in the multidomain nonribosomal peptide synthetase structure or standalone).

Bacteria↗
PubMed2001

Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.

The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.

ATP-Binding Cassette Transporters↗
PubMed2000

The McbB component of microcin B17 synthetase is a zinc metalloprotein.

The microcin B17 synthetase converts glycine, serine, and cysteine residues in a polypeptide precursor into oxazoles and thiazoles during the maturation of the Escherichia coli antibiotic Microcin B17. This multimeric enzyme is composed of three subunits (McbB, McbC, and McbD), and it employs both ATP and FMN as cofactors. The McbB subunit was purified as a fusion with the maltose-binding protein (MBP), and metal analysis revealed that this protein binds 0.91+/-0.17 zinc atoms. Upon incubation of MBP-McbB with excess zinc, the stoichiometry increased to two atoms of zinc bound, but metal binding to the second site resulted in a decrease in the heterocyclization activity when MBP-McbB was reconstituted with the other components of the synthetase. Apo-protein was prepared by using p-hydroxymercuriphenylsulfonic acid (PMPS), and loss of the metal caused a severe reduction in enzymatic activity. However, if dithiothreitol was added to the PMPS reactions within a few minutes, enzymatic activity was retained and MBP-McbB could be reconstituted with zinc. Spectroscopic analysis of the cobalt-containing protein and extended X-ray absorption fine structure analysis of the zinc-containing protein both provide evidence for a tetrathiolate coordination sphere. Site-directed mutants of MBP-McbB as well as the synthetase tagged with the calmodulin-binding peptide were constructed. Activity assays and metal analysis were used to determine which of the six cysteines in McbB are metal ligands. These results suggest that the zinc cofactor in McbB plays a structural role.

ATP-Binding Cassette Transporters↗
PubMed2000

Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains.

The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to nonribosomal peptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate ligase, VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), and VibH is a novel amide synthase that represents a free-standing condensation (C) domain. VibE and VibB are homologous to EntE and EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's ArCP domain. VibH then condenses this DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(1)-(2, 3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 min(-1) and a K(m) for acyl-VibB of 0.88 microM and for norspermidine of 1.5 mM. Exclusive monoacylation of a primary amine of norspermidine was observed. VibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, and hexylamine as substrates, albeit at lowered catalytic efficiencies. DHB-NSPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis. VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein-bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters.

Amide Synthases↗
PubMed2000

Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH.

Vibriobactin [N(1)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis[2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido]norspermidine] , is an iron chelator from the cholera-causing bacterium Vibrio cholerae. The six-domain, 270 kDa nonribosomal peptide synthetase (NRPS) VibF, a component of vibriobactin synthetase, has been heterologously expressed in Escherichia coli and purified. VibF has an unusual NRPS domain organization: cyclization-cyclization-adenylation-condensation-peptidyl carrier protein-condensation (Cy(1)-Cy(2)-A-C(1)-PCP-C(2)). VibF activates and covalently loads its PCP with L-threonine, and together with vibriobactin synthetase proteins VibE (adenylation) and VibB (aryl carrier protein) condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-VibF in the first demonstration of oxazoline formation by an NRPS cyclization domain. This enzyme-bound aryl oxazoline can be transferred by VibF to various amine acceptors but most efficiently to N(1)-(2, 3-dihydroxybenzoyl)norspermidine (k(cat) = 122 min(-1), K(m) = 1.7 microM), the product of 2,3-dihydroxybenzoyl-VibB, norspermidine, and VibH. This diacylated product undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed by VibF, to yield vibriobactin. Vibriobactin biosynthesis in vitro has thus been accomplished from four proteins, VibE, VibB, VibF, and VibH, with the substrates 2,3-dihydroxybenzoic acid, L-Thr, norspermidine, and ATP. Vibriobactin synthetase is an unusual NRPS in that all intermediates are not covalently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.

Catechols↗
PubMed2000

Acyl-CoA hydrolysis by the high molecular weight protein 1 subunit of yersiniabactin synthetase: mutational evidence for a cascade of four acyl-enzyme intermediates during hydrolytic editing.

Yersiniabactin (Ybt) synthetase is a three-subunit, 17-domain [7 domains in high molecular weight protein (HMWP)2, 9 in HMWP1, and 1 in YbtE] enzyme producing the virulence-conferring siderophore yersiniabactin in Yersinia pestis. The 350-kDa HMWP1 subunit contains a polyketide synthase module (KS-AT-MT(2)-KR-ACP) and a nonribosomal peptide synthetase module (Cy(3)-MT(3)-PCP(3)-TE). The full-length HMWP1 was heterologously overexpressed in Escherichia coli and purified to near homogeneity. The purified HMWP1 showed thioesterase activity toward acyl-CoAs, such as acetyl-CoA, benzoyl-CoA, and malonyl-CoA, with saturation kinetics and relative catalytic efficiencies of 172:50:1. A chain-releasing thioesterase (TE) activity is ascribed to the C-terminal TE domain, and this was substantiated by the fact that acyl-N-acetylcysteamines were hydrolyzed by the didomain PCP(3)-TE fragment of HMWP1. However, PCP(3)-TE failed to hydrolyze any of the acyl-CoAs, suggesting the TE domain does not recognize CoA moiety, thus the acyl-CoA hydrolysis by HMWP1 must involve other domains. Ser-to-Ala mutants in each of the AT, ACP, PCP(3), and TE domains reduced hydrolysis rates of the two fastest substrates, acetyl-CoA and benzoyl-CoA, by more than two orders of magnitude. Thus, the acyl-CoA hydrolysis activity requires 4 of the 9 domains of HMWP1, and it is consistent with autoacylation of the AT domain active site serine and subsequent passage of the itinerant acyl chain from AT to ACP to PCP(3) to the TE domain, a cascade of four sequential acyl-enzyme intermediates, for hydrolytic turnover. This could represent an editing pathway for this polyketide synthase/nonribosomal peptide synthetase assembly line.

Acyl Coenzyme A↗
PubMed2000

Deoxysugars in glycopeptide antibiotics: enzymatic synthesis of TDP-L-epivancosamine in chloroeremomycin biosynthesis.

The 2,3,6-trideoxysugar l-epivancosamine is the terminal sugar added to the aglycone scaffold in chloroeremomycin, a member of the vancomycin family of glycopeptide antibiotics. Five proteins from the chloroeremomycin biosynthetic cluster, ORF14 and ORF23 to ORF26, have been expressed heterologously in Escherichia coli and purified to near homogeneity, and each has been characterized for an enzymatic activity. These five enzymes reconstitute the complete biosynthesis of TDP-l-epivancosamine from TDP-4-keto-6-deoxy-d-glucose. This process involves C-2 deoxygenation, C-3 amination and methylation, C-5 epimerization, and C-4 ketoreduction. Intermediates and the final product of this pathway have been identified by mass spectrometry and NMR. The pathway established here represents the complete in vitro reconstitution of an unusual sugar for an important class of antibiotics and sets the groundwork for future combinatorial biosynthesis for new bioactive compounds.

Anti-Bacterial Agents↗
PubMed2000

Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase.

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.

Bacillus↗
PubMed2000

Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase.

The epimerase (E) domain of the three-domain (ATE) initiation module of Bacillus brevis gramicidin S synthetase equilibrates the Calpha configuration of the phenylalanyl moiety presented as Phe-S-4'-phosphopantetheine-modified (Ppant) acyl enzyme. Mutants at 22 residues of this E domain that are conserved across the approximately 450 residue E domains of nonribosomal peptide synthetases were constructed, and the PheATE derivatives expressed in Escherichia coli as C-terminal His tag fusions and then purified and assayed for three activities: (1) the L-Phe Calpha-[(3)H] exchange to solvent, (2) the rate of approach to D-Phe/L-Phe-S-Ppant acyl enzyme equilibrium from either L- or D-Phe, and (3) the rate of Phe-Pro dipeptidyl-S-Ppant enzyme formation with the downstream ProCAT module. We found that for wild-type PheATE epimerization is much faster than subsequent condensation, leading to a 1.9:1 ratio of D-Phe-S-Ppant/L-Phe-S-Ppant acyl enzyme. Only D-Phe is then transferred to yield D-Phe-L-Pro-S-Ppant ProCAT acyl enzyme. Among the mutants generated, three PheATE constructs, H753A, D757S, and Y976A, showed no detectable Calpha-(3)H washout, while E892A and R896A were among a larger set partially impaired. All these mutants were dramatically impaired in approach to D-Phe/L-Phe-S-Ppant equilibrium from either D- or L-Phe, while another construct, D767S, was asymmetrically impaired only for D-to-L-Phe direction. In the D-Phe-L-Pro dipeptidyl-S-Ppant condensation assay, the H753A and E892A forms of PheATE were only slightly active from L-Phe but unimpaired from D-Phe; N975A epimerizes faster than Y976A from L-Phe. When the chirality of the Phe-Pro-diketopiperazine released product was analyzed the D,L/L,L ratio from wild-type PheATE and ProCAT was 98:2. From E892A and N975A it was comparably 95:5 and 92:8, but H753A and Y976A yielded 56% of the L,L-product, reflecting a gain of function to transfer L-Phe. The 98:2 preference of wild-type PheATE for D-Phe transfer reflects the kinetically controlled stereopreference of the condensation (C) domain of ProCAT for the D-Phe-S-Ppant donor substrate. It may be that other NRPS C domains immediately downstream of E domains will likewise be D-selective.

Amino Acid Isomerases↗
PubMed2000

Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides.

BACKGROUND: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine. RESULTS: The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. CONCLUSIONS: Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.

Adenosine Diphosphate↗