New Advances in the Biosynthesis of Glycopeptide Antibiotics of the Vancomycin Type fromAmycolatopsis mediterranei

Süßmuth, Roderich D.; Pelzer, Stefan; Nicholson, Graeme; Walk, T.; Wohlleben, Wolfgang; Jung, Günther

doi:10.1002/(sici)1521-3773(19990712)38:13/14<1976::aid-anie1976>3.0.co;2-3

Cited by 82 publications

(66 citation statements)

References 4 publications

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“…The observation that L-and D-N-methylleucine are not adenylated by the A1 domain indicates that Leu methylation occurs after adenylation. This result is consistent with recent studies by Jung et al (18,19) with a strain of the balhimycin (Fig. 1) producer Amycolotopsis mediterranei in which the oxidative cross-linking genes were deleted, resulting in the production of a linear heptapeptide precursor.…”

Section: Resultssupporting

confidence: 93%

“…With respect to the timing of Leu L-to D-epimerization, Jung et al (19) found that the balhimycin heptapeptide precursor contained D-Leu (19), indicating that Leu epimerization occurs before oxidative cyclization, either at the stage of free Leu, Leu-S-PCP, peptide-S-PCP, or free linear peptide. There are two precedents that may be relevant to the CepA A1 initiation module selection and utilization of D-Leu.…”

Section: Resultsmentioning

confidence: 99%

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Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

Trauger¹

2000

Proceedings of the National Academy of Sciences

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The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates L-leucine and D-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with L-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with L-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

Trauger¹

2000

Proceedings of the National Academy of Sciences

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“…Recently, the gene cluster for balhimycin (4) biosynthesis has been identified in Amycolatopsis mediterranei DSM5908 by gene inactivation experiments (5), and the previously established genetic system (6) now permits the precise identification of gene functions and also the construction of new strains able to produce modified glycopeptides (5,(7)(8)(9).…”

mentioning

confidence: 99%

A Polyketide Synthase in Glycopeptide Biosynthesis

Pfeifer¹,

Nicholson²,

Ries³

et al. 2001

Journal of Biological Chemistry

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141

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Balhimycin, a vancomycin-type antibiotic from Amycolatopsis mediterranei, contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate-derived carbon backbone. After sequence analysis of the biosynthetic gene cluster, one gene, dpgA, for a predicted polyketide synthase (PKS) was identified, sharing 20 -30% identity with plant chalcone synthases. Inactivation of dpgA resulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon-like structure with three downstream genes that are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.

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“…This fact clearly points to a chlorination time point later than the stage of free ␤-OH-Tyr. Moreover, mutational studies revealed that chlorination occurs at a time point prior to oxidative cross-linking and subsequent glycosylation (94,125). Therefore, the timing of chlorination is proposed to occur after the ␤-OH-Tyr residues are tethered by thioester linkages to the respective PCPs of the NRPS assembly line.…”

Section: Halogenation Of Electron-rich Aromatic Rings and Unactivatedmentioning

confidence: 99%

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

Grünewald

Marahiel

2006

Microbiol Mol Biol Rev

200

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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New Advances in the Biosynthesis of Glycopeptide Antibiotics of the Vancomycin Type fromAmycolatopsis mediterranei

Cited by 82 publications

References 4 publications

Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

A Polyketide Synthase in Glycopeptide Biosynthesis

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

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