N-Methyltransferase function of the multifunctional enzyme enniatin synthetase

Billich, Andreas; Zocher, Rainer

doi:10.1021/bi00399a058

Cited by 65 publications

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“…Enniatin synthetase was the first N-methylcyclopeptide synthetase to characterized (4). Sequencing of the enniatin synthetase-corresponding gene (esyn1) from Fusarium scirpi revealed that the enzyme is one single polypeptide chain of 347 kDa (5).…”

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confidence: 99%

“…The 55-kDa N-methyltransferase portion M of the enzyme is located within the EB module. N-Methylation takes place after covalent binding of the amino acid on the surface of the corresponding peptide synthetase prior to peptide bond formation (4,6). S-Adenosyl-L-methionine (AdoMet) 1 serves as the methyl donor (4,7).…”

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confidence: 99%

“…N-Methylation takes place after covalent binding of the amino acid on the surface of the corresponding peptide synthetase prior to peptide bond formation (4,6). S-Adenosyl-L-methionine (AdoMet) 1 serves as the methyl donor (4,7). The mechanism of formation of N-methylated peptides has been elucidated in the case of the cyclodepsipeptides enniatin (8), beauvericin (9), and cyclosporin (10) and in actinomycin biosynthesis (11).…”

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“…Biochemical investigations of the N-methyltransferase function of enniatin synthetase (4) revealed that, similar to other methyltransferases, S-adenosyl-L-homocysteine (AdoHcy) and sinefungin are potent inhibitors of the AdoMet-dependent reaction. Sinefungin acted as a competitive inhibitor with respect to AdoMet, whereas AdoHcy exhibited an inhibition pattern characteristic for a partial competitive inhibitor, suggesting a discrete binding site for this inhibitor.…”

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Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

Hacker

Glinski

Hornbogen

et al. 2000

Journal of Biological Chemistry

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N-Methylated peptides like cyclosporins and enniatins constitute a class of pharmacologically interesting compounds. They are synthesized by a special class of enzymes representing hybrid systems of peptide synthetases and integrated Nmethyltransferase domains (1).Like other peptide synthetases, N-methylcyclopeptide synthetases follow a so-called thiol template mechanism, in which the substrate amino acids are activated as thioesters mediated by enzyme-bound 4Ј-phosphopantetheine residues (1-3). Enniatin synthetase was the first N-methylcyclopeptide synthetase to characterized (4). Sequencing of the enniatin synthetase-corresponding gene (esyn1) from Fusarium scirpi revealed that the enzyme is one single polypeptide chain of 347 kDa (5). It consists of the two modules EA and EB containing the two catalytic binding sites for the substrates D-hydroxyisovaleric acid and the branched-chain L-amino acid, respectively (5). The 55-kDa N-methyltransferase portion M of the enzyme is located within the EB module. N-Methylation takes place after covalent binding of the amino acid on the surface of the corresponding peptide synthetase prior to peptide bond formation (4, 6). S-Adenosyl-L-methionine (AdoMet) 1 serves as the methyl donor (4, 7). The mechanism of formation of N-methylated peptides has been elucidated in the case of the cyclodepsipeptides enniatin (8), beauvericin (9), and cyclosporin (10) and in actinomycin biosynthesis (11).Biochemical investigations of the N-methyltransferase function of enniatin synthetase (4) revealed that, similar to other methyltransferases, S-adenosyl-L-homocysteine (AdoHcy) and sinefungin are potent inhibitors of the AdoMet-dependent reaction. Sinefungin acted as a competitive inhibitor with respect to AdoMet, whereas AdoHcy exhibited an inhibition pattern characteristic for a partial competitive inhibitor, suggesting a discrete binding site for this inhibitor. Like other methyltransferases, the N-methyltransferase domain of enniatin synthetase can be affinity-labeled by UV irradiation in the presence of AdoMet labeled at the methyl group (4). The photoreaction was shown to be site-specific, and a binding stoichiometry of one methyl group/enzyme molecule was observed (4). It could be shown that AdoHcy diminished photolabeling of enniatin synthetase with [methyl-14 C]AdoMet or [methyl-3 H]AdoMet; but even in the presence of excess AdoHcy (100 M), it was not able to totally prevent the photoreaction, as did sinefungin; in contrast, the enzyme-bound radioactivity reached a reduced but constant level of 40% of the uninhibited control (4). This indicates that AdoHcy does not directly compete with AdoMet, but binds to a discrete inhibitory site. AdoHcy reduces the affinity of AdoMet for the enzyme, but, even at infinite high inhibitor concentrations, allows formation of an enzyme⅐AdoMet⅐AdoHcy complex, which still yields product. Furthermore, neither sinefungin nor AdoHcy affected substrate activation (adenylation and subsequent thioacylation). Interestingly, AdoHcy inhibited the formation of...

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Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

Hacker

Glinski

Hornbogen

et al. 2000

Journal of Biological Chemistry

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“…This hypothesis is supported by the finding that desmethyl enniatins are rarely found in fermentation broths, while they are produced during enniatin synthesis in vitro. 86 COD formation can be detected by a colorimetric picrate assay following extraction of mycelia, broth or assay mixtures, 88 or quantified by radioactively labeled SAM or amino acids. 84,86 COD analogs may be separated by TLC or HPLC.…”

Section: Reconstitution Of Cod Biosynthesis In Vitro With Purified Comentioning

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Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

et al. 2011

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Non‐ribosomal Biosynthesis of Linear and Cyclic Oligopeptides

Döhren

2002

Biopolymers Online

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Introduction Historical Introduction Proteins and Protein Domains in Non‐ribosomal Peptide Synthetase Systems Adenylate Domains Carrier Domains for the Transport of Intermediates Condensation Domains and Related Epimerization Domains Thioesterase Domains N‐methyltransferase Domains Oxidation and Reduction Domains The Reaction Cycle The Multi‐step Assembly Line Side Reactions Tailoring Reactions Isolation and Cloning of Biosynthetic Gene Clusters Occurrence and Detection of NRPS Clusters Structure of NRPS Clusters in Prokaryotes Eukaryotic NRPS Systems Manipulation of NRPS Systems Metabolic Engineering and Heterologous Expression Genetic Alterations of Programming Outlook and Perspectives

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N-Methyltransferase function of the multifunctional enzyme enniatin synthetase

Cited by 65 publications

References 12 publications

Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

Non‐ribosomal Biosynthesis of Linear and Cyclic Oligopeptides

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