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Mohamed A. Marahiel - One of the best experts on this subject based on the ideXlab platform.
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4 Phosphopantetheine transfer in primary and secondary metabolism of bacillus subtilis
Journal of Biological Chemistry, 2001Co-Authors: Henning D. Mootz, Robert Finking, Mohamed A. MarahielAbstract:Abstract 4′-Phosphopantetheine transferases (PPTases) transfer the 4′-Phosphopantetheine moiety of coenzyme A onto a conserved serine residue of acyl carrier proteins (ACPs) of fatty acid and polyketide synthases as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases. This posttranslational modification converts ACPs and PCPs from their inactive apo into the active holo form. We have investigated the 4′-phosphopantetheinylation reaction inBacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin. We identified and cloned ydcBencoding AcpS from B. subtilis, which complemented anEscherichia coli acps disruption mutant. B. subtilis AcpS and its substrate ACP were biochemically characterized. AcpS also modified the d-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that was not identified by the Bacillus genome project. On the other hand, Sfp was able to modify in vitro all acyl carrier proteins tested. We thereby extend the reported broad specificity of this enzyme to the homologous ACP. This in vitro cross-interaction between primary and secondary metabolism was confirmed under physiological in vivo conditions by the construction of a ydcB deletion in a B. subtilis sfp + strain. The genes coding for Sfp and its homolog Gsp from Bacillus brevis could also complement the E. coli acps disruption. These results call into question the essential role of AcpS in strains that contain a Sfp-like PPTase and consequently the suitability of AcpS as a microbial target in such strains.
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4′-Phosphopantetheine Transfer in Primary and Secondary Metabolism of Bacillus subtilis
The Journal of biological chemistry, 2001Co-Authors: Henning D. Mootz, Robert Finking, Mohamed A. MarahielAbstract:Abstract 4′-Phosphopantetheine transferases (PPTases) transfer the 4′-Phosphopantetheine moiety of coenzyme A onto a conserved serine residue of acyl carrier proteins (ACPs) of fatty acid and polyketide synthases as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases. This posttranslational modification converts ACPs and PCPs from their inactive apo into the active holo form. We have investigated the 4′-phosphopantetheinylation reaction inBacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin. We identified and cloned ydcBencoding AcpS from B. subtilis, which complemented anEscherichia coli acps disruption mutant. B. subtilis AcpS and its substrate ACP were biochemically characterized. AcpS also modified the d-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that was not identified by the Bacillus genome project. On the other hand, Sfp was able to modify in vitro all acyl carrier proteins tested. We thereby extend the reported broad specificity of this enzyme to the homologous ACP. This in vitro cross-interaction between primary and secondary metabolism was confirmed under physiological in vivo conditions by the construction of a ydcB deletion in a B. subtilis sfp + strain. The genes coding for Sfp and its homolog Gsp from Bacillus brevis could also complement the E. coli acps disruption. These results call into question the essential role of AcpS in strains that contain a Sfp-like PPTase and consequently the suitability of AcpS as a microbial target in such strains.
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Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases.
Chemistry & Biology, 1996Co-Authors: Torsten Stachelhaus, Anja Hüser, Mohamed A. MarahielAbstract:Abstract Background: A structurally diverse group of bioactive peptides is synthesized by peptide synthetases which act as templates for a growing peptide chain, attached to the enzyme via a thloester bond. The protein templates are composed of distinctive substrate-activating modules, whose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylation, thioester and peptide bond formation, as well as substrate modifications. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino aryl residues during non-ribosomal peptide synthesis, we have cloned and biochemically characterized that region of tyrocidine synthetase 1, TycA. Results: The 327-bp gene fragment encoding PCP was cloned using its homology to the genes for the acyl carrier proteins of fatty acid and polyketide biosynthesis. The protein was expressed as a His6, fusion protein, and purified in a single step by affinity chromatography. Incorporation of β-[3H]alanine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4′-Phosphopantetheine. When an adenylation domain is present to supply the amino adenylate moiety, PCP can be acylated in vitro. Conclusions: PCP can bind covalently to the cofactor Phosphopantetheine and can subsequently be acylated, strongly supporting the multiple carrier model of non-ribosomal peptide synthesis. The adenylation and thiolation domains can each act as independent multifunctional enzymes, further confirming the modular structure of peptide synthees, and can also perform sequential steps in trans, as do multienzyme complexes.
Tina Izard - One of the best experts on this subject based on the ideXlab platform.
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substrate induced asymmetry and channel closure revealed by the apoenzyme structure of mycobacterium tuberculosis Phosphopantetheine adenylyltransferase
Protein Science, 2004Co-Authors: Van K Morris, Tina IzardAbstract:Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in prokaryotic coenzyme A (CoA) biosynthesis, directing the transfer of an adenylyl group from ATP to 4'-Phosphopantetheine (Ppant) to yield dephospho-CoA (dPCoA). The crystal structures of Escherichia coli PPAT bound to its substrates, product, and inhibitor revealed an allosteric hexameric enzyme with half-of-sites reactivity, and established an in-line displacement catalytic mechanism. To provide insight into the mechanism of ligand binding we solved the apoenzyme (Apo) crystal structure of PPAT from Mycobacterium tuberculosis. In its Apo form, PPAT is a symmetric hexamer with an open solvent channel. However, ligand binding provokes asymmetry and alters the structure of the solvent channel, so that ligand binding becomes restricted to one trimer.
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Substrate‐induced asymmetry and channel closure revealed by the apoenzyme structure of Mycobacterium tuberculosis Phosphopantetheine adenylyltransferase
Protein science : a publication of the Protein Society, 2004Co-Authors: Van K. Morris, Tina IzardAbstract:Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in prokaryotic coenzyme A (CoA) biosynthesis, directing the transfer of an adenylyl group from ATP to 4'-Phosphopantetheine (Ppant) to yield dephospho-CoA (dPCoA). The crystal structures of Escherichia coli PPAT bound to its substrates, product, and inhibitor revealed an allosteric hexameric enzyme with half-of-sites reactivity, and established an in-line displacement catalytic mechanism. To provide insight into the mechanism of ligand binding we solved the apoenzyme (Apo) crystal structure of PPAT from Mycobacterium tuberculosis. In its Apo form, PPAT is a symmetric hexamer with an open solvent channel. However, ligand binding provokes asymmetry and alters the structure of the solvent channel, so that ligand binding becomes restricted to one trimer.
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Rhombohedral crystals of Mycobacterium tuberculosis Phosphopantetheine adenylyltransferase.
Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: Kathrin L Brown, Van K Morris, Tina IzardAbstract:The penultimate step of prokaryotic coenzyme A (CoA) biosynthesis is directed by the essential enzyme Phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3), an attractive target for antibiotics. The reaction catalyzed by PPAT is rate-limiting and involves the transfer of an adenylyl group from ATP to 4'-Phosphopantetheine to form 3'-dephospho-CoA. Rhombohedral crystals of PPAT from Mycobacterium tuberculosis (Rv2965c) were obtained. The crystals belong to space group R32, with unit-cell parameters a = 68.69 A, alpha = 91.81 degrees. The crystals diffract to better than 2 A resolution on a Cu Kalpha rotating-anode generator. The packing density for one polypeptide chain in the asymmetric unit is 2.89 A(3) Da(-1), with a solvent content of 0.57.
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A novel adenylate binding site confers Phosphopantetheine adenylyltransferase interactions with coenzyme A.
Journal of bacteriology, 2003Co-Authors: Tina IzardAbstract:Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway. PPAT catalyzes the reversible transfer of an adenylyl group from Mg2+:ATP to 4′-Phosphopantetheine to form 3′-dephospho-CoA (dPCoA) and pyrophosphate. The high-resolution crystal structure of PPAT complexed with CoA has been determined. Remarkably, CoA and the product dPCoA bind to the active site in distinct ways. Although the phosphate moiety within the Phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites. Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme. Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion.
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The crystal structures of Phosphopantetheine adenylyltransferase with bound substrates reveal the enzyme's catalytic mechanism.
Journal of molecular biology, 2002Co-Authors: Tina IzardAbstract:Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in the coenzyme A pathway that catalyzes the reversible transfer of an adenylyl group from ATP to 4′-Phosphopantetheine (Ppant) in the presence of magnesium. To investigate the reaction mechanism, the high-resolution crystal structures of the Escherichia coli PPAT have been determined in the presence of either ATP or Ppant. Structural details of the catalytic center revealed specific roles for individual amino acid residues involved in substrate binding and catalysis. The side-chain of His18 stabilizes the expected pentacovalent intermediate, whereas the side-chains of Thr10 and Lys42 orient the nucleophile for an in-line displacement mechanism. The binding site for the manganese ion that interacts with the phosphate groups of the nucleotide has also been identified. Within the PPAT hexamer, one trimer is in its substrate-free state, whereas the other is in a substrate-bound state.
Se Won Suh - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of Phosphopantetheine adenylyltransferase from Enterococcus faecalis in the ligand-unbound state and in complex with ATP and pantetheine
Molecules and Cells, 2011Co-Authors: Hye-jin Yoon, Ji Yong Kang, Hyung Ho Lee, Bunzo Mikami, Se Won SuhAbstract:Phosphopantetheine adenylyltransferase (PPAT) catalyzes the reversible transfer of an adenylyl group from ATP to 4′-Phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate in the Coenzyme A (CoA) biosynthetic pathway. Importantly, PPATs are the potential target for developing antibiotics because bacterial and mammalian PPATs share little sequence homology. Previous structural studies revealed the mechanism of the recognizing substrates and products. The binding modes of ATP, ADP, Ppant, and dPCoA are highly similar in all known structures, whereas the binding modes of CoA or 3′-phosphoadenosine 5′-phosphosulfate binding are novel. To provide further structural information on ligand binding by PPATs, the crystal structure of PPAT from Enterococcus faecalis was solved in three forms: (i) apo form, (ii) binary complex with ATP, and (iii) binary complex with pantetheine. The substrate analog, pantetheine, binds to the active site in a similar manner to Ppant. The new structural information reported in this study including pantetheine as a potent inhibitor of PPAT will supplement the existing structural data and should be useful for structurebased antibacterial discovery against PPATs.
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Overexpression, crystallization and preliminary X-ray crystallographic analysis of Phosphopantetheine adenylyltransferase from Enterococcus faecalis.
Acta crystallographica. Section F Structural biology and crystallization communications, 2006Co-Authors: Ji Yong Kang, Hyung Ho Lee, Hye-jin Yoon, Hyoun Sook Kim, Se Won SuhAbstract:Phosphopantetheine adenylyltransferase, an essential enzyme in the coenzyme A biosynthetic pathway, catalyzes the reversible transfer of an adenylyl group from ATP to 4'-Phosphopantetheine, yielding 3'-dephospho-CoA and pyrophosphate. Enterococcus faecalis PPAT has been overexpressed in Escherichia coli as a fusion with a C-terminal purification tag and crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium HEPES pH 7.5, 0.8 M sodium dihydrogen phosphate and 0.8 M potassium dihydrogen phosphate. X-ray diffraction data were collected to 2.70 A at 100 K. The crystals belong to the primitive tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = b = 160.81, c = 225.68 A. Four copies of the hexameric molecule are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (V(M)) of 3.08 A(3) Da(-1) and a solvent content of 60.1%.
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Crystallization and preliminary X-ray crystallographic studies of Phosphopantetheine adenylyltransferase from Helicobacter pylori.
Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: Su Jeong Eom, Hyung Jun Ahn, Hyung Wook Kim, Seung-hun Baek, Se Won SuhAbstract:Phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) is an essential enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the reversible transfer of an adenylyl group from ATP to 4'-Phosphopantetheine to form 3'-dephospho-CoA. PPAT from Helicobacter pylori has been overexpressed in Escherichia coli and crystallized at 296 K using sodium chloride as a precipitant by the hanging-drop vapour-diffusion method. X-ray diffraction data have been collected to 2.00 A resolution at 100 K using synchrotron radiation. The crystals belong to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 80.50, c = 143.05 A, alpha = beta = 90, gamma = 120 degrees. Six monomers of PPAT are likely to be present in the asymmetric unit, giving a V(M) of 2.39 A(3) Da(-1) and a solvent content of 49%.
Torsten Stein - One of the best experts on this subject based on the ideXlab platform.
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gramicidin s synthetase 1 phenylalanine racemase a prototype of amino acid racemases containing the cofactor 4 Phosphopantetheine
Biochemistry, 1995Co-Authors: Torsten Stein, Britta Kluge, Joachim Vater, Peter Franke, Albrecht Otto, Brigitte WittmannlieboldAbstract:: The biosynthesis of the decapeptide antibiotic gramicidin S in Bacillus brevis ATCC 9999 is catalyzed by a multienzyme system consisting of two multifunctional proteins, gramicidin S synthetase 1 and 2, encoded by the grsA and grsB genes, respectively. Gramicidin S synthetase 1 (phenylalanine racemase, EC 5.1.1.11, GS1) racemizes phenylalanine in the thioester-bound stage. The amount of 4'-Phosphopantetheine liberated from highly purified GS1 was determined microbiologically using Lacto-bacillus plantarum as the test organism. It matches exactly with the amount of L-[14C]phenylalanine covalently incorporated by GS1 as thioester. The reaction center of GS1 for L-phenylalanine thiolation and racemization was labeled with [3H]iodoacetic acid. After tryptic fragmentation of the 3H-carboxymethylated enzyme, the active site peptide for thioester binding and racemization of phenylalanine was isolated in pure form by multistep methodology and investigated by sequence, amino acid, and mass spectrometric analysis. A 4'-Phosphopantetheine carrier was found to be attached to the active site serine of the consensus motif LGGDSI forming the thiolation site of phenylalanine. These specific properties establish GS1 as a prototype of amino acid racemases using 4'-Phosphopantetheine as a cofactor and yield further evidence that multiple Pan carriers are involved in gramicidin S formation. Our results are strong evidence for the "multiple carrier model" as a new concept of nonribosomal peptide biosynthesis at protein templates as recently proposed [Stein, T., et al. (1994) FEBS Lett. 340, 39-44].
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Detection of 4'-Phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2.
FEBS letters, 1994Co-Authors: Torsten Stein, Joachim Vater, Peter Franke, Volker Kruft, Brigitte Wittmann-liebold, Maria Panico, Roy Mc Dowell, Howard R. MorrisAbstract:Biosynthesis of gramicidinS in Bacillus brevis is catalysed by a multienzyme system consisting of two multifunctional proteins, gramicidinS synthetase 1 and 2 codified by the grsA and grsB genes, respectively. GramicidinS synthetase 2 shows a modular architecture of four amino acid-activating domains each containing a thioester binding motif LGG H/D S L/I highly conserved in its C-terminal region, as demonstrated by sequence analysis of the grsB gene [W. Schlumbohm et al. (1991) J. Biol. Chem. 266, 23135-23141]. This multienzyme was specifically labeled at the thioester binding site of l-valine with [3H]N-ethylmaleimide using a substrate protection technique. After enzymatic digestion a labeled active site peptide was isolated in pure form by multistep methodology. This fragment was identified by gas-phase sequencing as the active site peptide of the thiotemplate site for l-Val by comparison with the grsB gene sequence. By mass spectrometry in combination with amino acid analysis it was demonstrated that a 4'-Phosphopantetheine carrier was attached to the active serine in this motif. Our results give evidence that multiple peripheral 4'-Phosphopantetheine carriers are involved in the formation of gramicidinS in contrast to a central carrier arm as assumed in the original version of the thiotemplate mechanism. A ‘Multiple Carrier Model’ of nonribosomal peptide biosynthesis is proposed.
Arie Geerlof - One of the best experts on this subject based on the ideXlab platform.
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Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli
The Journal of biological chemistry, 1999Co-Authors: Arie Geerlof, Ann Lewendon, William V. ShawAbstract:Abstract Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4′-Phosphopantetheine yielding 3′-dephospho-CoA and pyrophosphate. Wild-type PPAT fromEscherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamedcoaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3′-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.
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The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity
The EMBO journal, 1999Co-Authors: Tina Izard, Arie GeerlofAbstract:Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4'-Phosphopantetheine, yielding dephospho-CoA (dPCoA). Each Phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide-binding fold that is structurally similar to that in class I aminoacyl-tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl-tRNA synthetases suggests nucleophilic attack by the 4'-Phosphopantetheine on the alpha-phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice-like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence-binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.