Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides

Schauwecker, Florian; Pfennig, Frank; Grammel, Nicolas; Keller, Ullrich

doi:10.1016/s1074-5521(00)00103-4

Cited by 68 publications

(62 citation statements)

References 34 publications

(67 reference statements)

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“…Furthermore, the corresponding sequences of bacterial synthetases are given: microcystin synthetase A from Microcystis sp. (MCYA_mm1) (30), actinomycin synthetase II from Streptomyces chrysomallus (ACMC_sm2 and ACMC_sm3) (31), and pristinamycin synthetase from Streptomyces pristinaespiralis (SNBDEpri) and Streptomyces virginiae (SNBDEvir) (32). The N-methyltransferase domains of the enniatin synthetases of Fusarium share high similarity (50 -60%) with the other N-methyltransferase domains of eucaryotic origin (cyclosporin synthetase), but lower similarity (20 -25%) with the procaryotic systems.…”

Section: Sequence Analysis Of N-methyltransferase Domains Of Peptide mentioning

confidence: 99%

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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Section: Sequence Analysis Of N-methyltransferase Domains Of Peptide mentioning

confidence: 99%

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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“…The modular organization of protein domains in NRPSs has stimulated efforts to swap genes encoding heterologous domains into NRPSs with the goal of producing nonnatural variants of the original small-molecule product (3,4). Although limited success has been achieved with the production of nonribosomal peptide (NRP) variants (5)(6)(7)(8)(9)(10)(11)(12)(13)(14), the resultant chimeric NRPSs are usually nonfunctional or heavily impaired, often yielding too little of the new product to make production feasible. It is not yet known why chimeric NRPSs suffer large reductions in activity, although it is likely that domain swapping disrupts quaternary interactions between protein domains in the assembly line (15).…”

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confidence: 99%

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

Fischbach

Lai

Roche

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

135

107

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Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with Ϸ10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/ polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (10 3 to 10 4 clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts.nonribosomal peptide ͉ polyketide

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“…1) (135), pristinamycin (Fig. 1) (24, 80), and actinomycin (102), contain N-methylated peptide bonds. In most cases, N-methylation is introduced by an in cis-acting N-methyltransferase (ϳ420 aa) which is inserted into the C-terminal end of the accompanying A domain.…”

Section: C-and N-methylation Of Nonribosomal Peptidesmentioning

confidence: 99%

“…In most cases, N-methylation is introduced by an in cis-acting N-methyltransferase (ϳ420 aa) which is inserted into the C-terminal end of the accompanying A domain. Transfer of the S-methyl group of SAM to the ␣-amino group occurs when the respective amino acid is tethered to the ppan cofactor, whereupon amide bond formation can occur, generating an N-methylated peptide bond (102). However, in contrast to in cis-acting N-methyltransferases, MtfA of the chloroeremomycin biosynthetic system catalyzes in trans methyl transfer to the N-terminal leucine of chloroeremomycin (19).…”

Section: C-and N-methylation Of Nonribosomal Peptidesmentioning

confidence: 99%

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

Grünewald

Marahiel

2006

Microbiol Mol Biol Rev

202

134

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SUMMARY Non-ribosomally synthesized peptides have compelling biological activities ranging from antimicrobial to immunosuppressive and from cytostatic to antitumor. The broad spectrum of applications in modern medicine is reflected in the great structural diversity of these natural products. They contain unique building blocks, such as d-amino acids, fatty acids, sugar moieties, and heterocyclic elements, as well as halogenated, methylated, and formylated residues. In the past decades, significant progress has been made toward the understanding of the biosynthesis of these secondary metabolites by nonribosomal peptide synthetases (NRPSs) and their associated tailoring enzymes. Guided by this knowledge, researchers genetically redesigned the NRPS template to synthesize new peptide products. Moreover, chemoenzymatic strategies were developed to rationally engineer nonribosomal peptides products in order to increase or alter their bioactivities. Specifically, chemical synthesis combined with peptide cyclization mediated by nonribosomal thioesterase domains enabled the synthesis of glycosylated cyclopeptides, inhibitors of integrin receptors, peptide/polyketide hybrids, lipopeptide antibiotics, and streptogramin B antibiotics. In addition to the synthetic potential of these cyclization catalysts, which is the main focus of this review, different enzymes for tailoring of peptide scaffolds as well as the manipulation of carrier proteins with reporter-labeled coenzyme A analogs are discussed.

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Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides

Cited by 68 publications

References 34 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

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

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