Two multifunctional peptide synthetases and an O-methyltransferase are involved in the biosynthesis of the DNA-binding antibiotic and antitumour agent saframycin Mx1 from Myxococcus xanthus

Pospiech, Andreas; Bietenhader, Jürg; Schupp, Thomas

doi:10.1099/00221287-142-4-741

Cited by 91 publications

(65 citation statements)

References 36 publications

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“…Previous studies on SFM-Mx1 and SAC-B biosynthesis revealed a relatively rare reductase (RE) domain that contains a NAD(P)H binding site at the C-terminal ends of SafA and SacC (33,46), both of which are NRPSs involved in the tetrapeptidyl backbone formation. These RE domains may act on the PCP-tethered polypeptidyl intermediate and reductively release it from the PCP as a linear aldehyde (21), instead of the thioesterase (TE) functionality at the C-terminal ends of typical NRPSs.…”

Section: Resultsmentioning

confidence: 99%

“…Based on the colinearity rule (25), wherein the NRPS module organization parallels the order of the amino acid residues in the resultant polypeptide, sequential incorporation of Ala, Gly, and two Tyr derivatives into the tetrapeptide in SAM-Mx1 biosynthesis was previously speculated to be directed by four successive modules (i.e., SafB-AL-PCP0, SafB-C1-A1-PCP1, SafA-C2-A2-PCP2, and SafA-C3-A3-PCP3-RE) (Fig. 6B) (33). Since SacA in SAC-B biosynthesis lacks the first module AL-PCP0, a bifunctional adenylation activation by SacA or direct incorporation of an Ala-Gly dipeptide into the tetrapeptide by SacA was hypothesized ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1. The partial biosynthetic gene cluster of SFM-Mx1 (with a hydroquinone form of the E ring, a hydroxy group at the C-21 position, and a reserved ␣-amino group of Ala in comparison to SFM-A) and the entire biosynthetic gene cluster of SAC-B (one of the structurally simplest members in the SFM family) were cloned from Myxococcus xanthus in 1995 (32,33) and Pseudomonas fluorescens in 2005 (46), respectively, indeed revealing a nonribosomal peptide synthetase (NRPS) system for the formation of an identical tetrapeptide intermediate. In both cases, sequential incorporation of Ala, Gly, and Tyr derivatives into the backbone was speculated to be catalyzed by NRPSs in a colinear way according to the substrate specificity of the NRPS modules.…”

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confidence: 99%

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Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

et al. 2008

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Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

et al. 2008

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“…This reductive mechanism terminating nonribosomal peptide biosynthesis has been described for myxochelin in Stigmatella aurantiaca Sg a15 (Gaitatzis et al, 2001), and for saframycin Mx1 in Myxococcus xanthus (Pospiech et al, 1996).…”

Section: Formation Of the CM Hydroxymethyl Groupmentioning

confidence: 99%

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

2004

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Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1 H-NMR established that the principal metabolite in the disrupted strains was the a-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH 2 -dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

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“…29,30 To date, three biosynthetic gene clusters of tetrahydroisoquinoline antibiotics 14, safracin B ( Figure 5, 16) and saframycin Mx1 (17) have been identified and their bioinfomatic analysis indicated that a saframycin backbone is constructed by NRPS. 30,31,32 Recently, Wen and coworkers proposed that the saframycin NRPS (SfmA, SfmB, SfmC) unusually assembles the core skeleton via a putative tetrapeptidyl intermediate using SfmC in an iterative manner. 32 However, detailed biosynthetic mechanism of this unusual transformation has not been uncovered.…”

Section: Enzymatic Synthesis Of Core Skeleton Of Antitumor Antibioticmentioning

confidence: 99%

Enzymatic Synthesis of Molecular Skeletons of Complex Antitumor Antibiotics with Non-ribosomal Peptide Synthetases

Watanabe

Oguri

Oikawa

2009

J. Synth. Org. Chem. Jpn.

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Nonribosomal peptide synthetase (NRPS) is a programmable modular machinery which produces a number of biologically active small-molecule peptides. Recent progress on understanding the catalytic mechanism of NRPS enabled us to engineer this complex catalytic system. We demonstrated both in vivo and in vitro enzymatic synthesis of complex natural antitumor peptides echinomycin and saframycin. An Escherichia coli-based expression system served as a flexible yet robust platform for producing complex natural products and their analogs by deletion, mutation and swapping of a specific subunit. The excised thioesterase domain of echinomycin exhibited remarkable substrate tolerance and created a cyclic peptide library. On the other hand, saframycin NRPS catalyzed highly unusual seven-step transformations to construct a pentacyclic tetrahydro-isoquinoline skeleton. According to the generally accepted mechanism (colinearity rule), 3 NRPS produces polypeptides different in size and amino acid-sequence with various modifications. An important strategy that this enzyme adopts is that condensation substrates are always bound to the enzyme (T) and that the chain-elongated intermediates are never released from NRPS until the final cleavage. Therefore, this system guarantees reliable and safe delivery of the substrates. This allows rather broad substrate specificity of the C-domain. Each module is a self-consistent portable component. This suggests that replacing A-domain or a specific module may create new peptides in a programmable manner. Hence, the rational design of NRPS makes it possible to synthesize novel peptides just in the same way as organic synthesis. In this account, we describe an enzymatic synthesis of two important families of antitumor peptide antibiotics, echinomycin and saframycin. Enzymatic Synthesis of Echinomycin and its DerivativesEchinomycin (1) belongs to quinomycin bis-intercalator antibiotics (Figure 2). 4 This class of antibiotics are dimeric cyclic peptides with a pair of chromophores and have potent DNA binding affinities at a level between nM to M with different sequence selectivities. Reflecting these distinct affinities, bis-intercalator antibiotics such as 1-4 show fairly different inhibitory activities against various enzymes such as DNA polymerase, RNA polymerase, reverse transcriptase and topoisomerase. Based on structure-activity relationships among this class of antibiotics, one can expect that minute structural difference may drastically change profiles between antitumor activity and toxicity. To examine this possibility, we became interested in synthesizing echinomycin derivatives.To identify and isolate the echinomycin biosynthetic gene cluster, a cosmid library was constructed using S. lasaliensis total DNA. Screening with the PCR product from degenerate primers for NRPS afforded plasmids encoding the 36-kb echinomycin biosynthetic gene clusters. 5 Homology of these genes enabled us to speculate their functions in the biosynthesis of quinoxaline-2-carboxylic acid (5, QXC) and ...

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Two multifunctional peptide synthetases and an O-methyltransferase are involved in the biosynthesis of the DNA-binding antibiotic and antitumour agent saframycin Mx1 from Myxococcus xanthus

Cited by 91 publications

References 36 publications

Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

Enzymatic Synthesis of Molecular Skeletons of Complex Antitumor Antibiotics with Non-ribosomal Peptide Synthetases

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