An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

Schlumbohm, Wilhelm; Stein, Torsten; Ullrich, Christian; Vater, Joachim; Krause, Michael; Marahiel, Mohamed A.; Kruft, Volker; Wittmann‐Liebold, Brigitte

doi:10.1016/s0021-9258(18)54473-2

Cited by 132 publications

(36 citation statements)

References 53 publications

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“…Correspondingly, in the activation of other residues catalyzed by ACMS II, individual 4'phosphopantetheine residues would be involved not only as acceptors but also as carriers of individual partial sequences of the actinomycin peptide chain. This conclusion is supported by recent data concerning the sequence of an active-site peptide fragment of the valine binding domain of gramicidin synthetase II (Schlumbohm et al, 1991;Turgay et al, 1992), which indicated the presence of a serine instead of cysteine in the catalytic centers of various peptide synthetases. These serines most probably represent binding sites for 4'-phosphopantetheine (Marahiel, 1992).…”

Section: Resultssupporting

confidence: 65%

Epimerization of the D-Valine Portion in the Biosynthesis of Actinomycin D

Stindl¹,

Keller²

1994

Biochemistry

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In the biosynthesis of actinomycin, the multifunctional actinomycin synthetase II (ACMS II) assembles 4-methyl-3-hydroxyanthranilic acid (4-MHA), L-threonine and D-valine, the first three residues of the 4-MHA peptide lactone chain. ACMS II activates L-threonine and L-valine but not D-valine as thioesters via their adenylates, and there is no epimerization of the covalently bound L-valine. When L-threonine and L-valine are presented to the enzyme together with the 4-MHA analogue p-toluic acid and the 4-MHA-activating enzyme ACMS I, ACMS II forms the two diastereomers p-toluyl-L-Thr-L-Val and p-toluyl-L-Thr-D-Val in equal amounts along with p-toluyl-L-Thr in a cofactor-independent manner. Studies with [2,3-3H2]valine revealed that p-toluyl-L-Thr-D-Val contained approximately 50% of the tritium label found in the LL-diastereomer. Concomitantly, radioactive water was formed due to enzyme-catalyzed hydrogen exchange with the solvent during epimerization. In the absence of threonine (or MgATP), however, the amount of radioactive water formed from [3H]valine was significantly less, which suggests that the peptide bond between L-threonine and L-valine is formed prior to the epimerization at C-2 of valine. The facts that both LL- and LD-acyldipeptides are equally present on the enzyme's surface--as revealed by using 14C-labeled threonine or valine as precursors--and that the L-valine in the LL-diastereomer apparently has not lost hydrogen strongly suggests that the LL-diastereomer is an obligatory intermediate in the formation of the LD-dipeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 65%

Epimerization of the D-Valine Portion in the Biosynthesis of Actinomycin D

Stindl¹,

Keller²

1994

Biochemistry

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“…LGG(H,D)SL, shared by acyl carrier proteins (ACP) of fatty acid synthetases (Schlumbohm et al, 1991;Stein et al, 1995), has been found positionally conserved in all peptide synthetase modules, suggesting the aminoacylation site to be the cysteamine moiety of the cofactor. This motif also strongly resembles sites from polyketide-forming enzyme systems known to carry 4/-phosphopantetheine as cofactor (van Liempt et al, 1991).…”

mentioning

confidence: 99%

Characterization of tyrocidine synthetase 1 (TY1): Requirement of posttranslational modification for peptide biosynthesis

Pfeifer¹,

Pavela-Vrančić²,

H³

et al. 1995

Biochemistry

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Tyrocidine synthetase 1 (TY1), produced by Bacillus brevis ATCC 8185, consists of a single multifunctional polypeptide chain catalyzing the activation, thioesterification, and epimerization of phenylalanine. Because we were concerned about possible posttranslational issues, a comparative study between the wild-type isolate and the in Escherichia coli overexpressed protein was performed. Analysis by matrix assisted laser desorption mass spectrometry (MALDI) provided a molecular mass of 122,516 +/- 120 Da for the recombinant protein, which is in agreement with the value of 122,590 Da calculated from the gene sequence. MALDI analysis of the tryptic fragments revealed that in the recombinant TY1 the putative 4'-phosphopantetheine binding site (562Ser) is not modified by the cofactor. The substrate specificity profiles of the amino acid dependent ATP[32P]PPi exchange reactions were identical, including activation of L-phenylserine, L-tyrosine, and L-methionine. However, the rates of the reverse adenylation reaction for the recombinant protein were only 22% relative to those of the wild-type enzyme. The aminoacylation levels of about 60% for TY1 from Bacillus brevis reduced to 1.4% in the overexpressed protein. A similar distribution of the D- and the L-isomer was detected at the thioester attachment site. The pI values of the wild-type and expressed TY1 are 4.9 and 5.0, respectively. In conclusion, it could be established that apo- and holo-TY1 differ in their amino acid activating properties. Posttranslational modification by 4'-phosphopantetheine is an essential requirement for aminoacylation, epimerization, and thus the functioning of the multienzyme in peptide synthesis.

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“…Carrier proteins are four helix bundles of B80 amino acids, found either as isolated proteins or as domains in larger multi-domain proteins, such as polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). 3 They share a conserved serine residue that is post-translationally modified with a phosphopantetheine linker. 4 P450s that oxidise CP-bound substrates can currently be split into four classes (Fig.…”

Section: Cytochrome P450s and Carrier Proteinsmentioning

confidence: 99%

Carrier protein substrates in cytochrome P450-catalysed oxidation

Cryle

2011

Metallomics

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Enzymes of the cytochrome P450 superfamily can catalyse many types of oxidative transformations of a diverse substrate range. This review summarises recent work on an subset of P450s that accept their substrates in carrier protein-bound form. These examples show how the oxidative power and precision that P450s bring to natural products biosynthesis can be coupled with the advantages of using a carrier protein as a scaffold for oxidation.

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An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

Cited by 132 publications

References 53 publications

Epimerization of the D-Valine Portion in the Biosynthesis of Actinomycin D

Epimerization of the D-Valine Portion in the Biosynthesis of Actinomycin D

Characterization of tyrocidine synthetase 1 (TY1): Requirement of posttranslational modification for peptide biosynthesis

Carrier protein substrates in cytochrome P450-catalysed oxidation

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