Aminocoumarin Moiety Research Articles

Two transcription factors, CabA and CabR, are independently involved in multilevel regulation of the biosynthetic gene cluster encoding the novel aminocoumarin, cacibiocin.

Aminocoumarins are potent antibiotics belonging to a relatively small group of secondary metabolites produced by actinomycetes. Genome mining of Catenulispora acidiphila has recently led to the discovery of a gene cluster responsible for biosynthesis of novel aminocoumarins, cacibiocins. However, regulation of the expression of this novel gene cluster has not yet been analyzed. In this study, we identify transcriptional regulators of the cacibiocin gene cluster. Using a heterologous expression system, we show that the CabA and CabR proteins encoded by cabA and cabR genes in the cacibiocin gene cluster control the expression of genes involved in the biosynthesis, modification, regulation, and potentially, efflux/resistance of cacibiocins. CabA positively regulates the expression of cabH (the first gene in the cabHIYJKL operon) and cabhal genes encoding key enzymes responsible for the biosynthesis and halogenation of the aminocoumarin moiety, respectively. We provide evidence that CabA is a direct inducer of cacibiocin production, whereas the second transcriptional factor, CabR, is involved in the negative regulation of its own gene and cabT-the latter of which encodes a putative cacibiocin transporter. We also demonstrate that CabR activity is negatively regulated in vitro by aminocoumarin compounds, suggesting the existence of analogous regulation in vivo. Finally, we propose a model of multilevel regulation of gene transcription in the cacibiocin gene cluster by CabA and CabR.

ChemInform Abstract: New Aminocoumarins from the Rare Actinomycete Catenulispora acidiphila DSM 44928: Identification, Structure Elucidation, and Heterologous Production.

Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L(-1) in C. acidiphila to 60 mg L(-1) in S. coelicolor M1152. By HR-MS and NMR analysis, cacibiocin A was found to contain a 3-amino-4,7-dihydroxycoumarin moiety linked by an amide bond to a pyrrole-2,5-dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6' and 8' of the aminocoumarin moiety.

New Aminocoumarins from the Rare ActinomyceteCatenulispora acidiphilaDSM 44928: Identification, Structure Elucidation, and Heterologous Production

Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L(-1) in C. acidiphila to 60 mg L(-1) in S. coelicolor M1152. By HR-MS and NMR analysis, cacibiocin A was found to contain a 3-amino-4,7-dihydroxycoumarin moiety linked by an amide bond to a pyrrole-2,5-dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6' and 8' of the aminocoumarin moiety.

Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis

MbtH-like proteins consist of ∼70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes.

Adenylate‐Forming Enzymes of Rubradirin Biosynthesis: RubC1 Is a Bifunctional Enzyme with Aminocoumarin Acyl Ligase and Tyrosine‐Activating Domains

The biosynthesis of aminocoumarin antibiotics requires two acyladenylate-forming enzymes: one for the activation of L-tyrosine as a precursor of the aminocoumarin moiety and another for the linkage of an acyl moiety to the aminocoumarin moiety. Unexpectedly, the biosynthetic gene cluster of the aminocoumarin antibiotic rubradirin was found to contain three genes for putative acyladenylate-forming enzymes of aminocoumarin biosynthesis and conjugation. We expressed, purified, and investigated these three proteins. Orf4 (55 kDa) was shown to be an active aminocoumarin acyl ligase. RubF6 (56 kDa) was inactive, but could be converted into an active enzyme by site-directed mutagenesis. RubC1 (138 kDa) was shown to be a unique bifunctional enzyme, comprising an aminocoumarin acyl ligase, and tyrosine-adenylation and peptidyl-carrier domains. This natural hybrid enzyme is unique among known proteins. A hypothesis is proposed as to how such an enzyme could offer a particularly effective machinery for aminocoumarin antibiotic biosynthesis.

Structures of the TetR-like Simocyclinone Efflux Pump Repressor, SimR, and the Mechanism of Ligand-Mediated Derepression

Simocyclinone D8 (SD8), a potent DNA gyrase inhibitor made by Streptomyces antibioticus, is exported from the producing organism by the SimX efflux pump. The expression of simX is under the control of SimR, a member of the TetR family of transcriptional regulators. SimR represses simX transcription by binding to operators in the intergenic region between simR and simX. Previously, we have shown that the mature antibiotic SD8 or its biosynthetic intermediate, simocyclinone C4, can dissociate SimR from its operators, leading to derepression of simX and export of SD8 from the cell. This provides a mechanism that couples the biosynthesis of the antibiotic to its export. Here, we report the crystal structures of SimR alone and in complex with either SD8 or simocyclinone C4. The ligand-binding pocket is unusual compared to those of other characterized TetR-family transcriptional regulators: the structures show an extensive ligand-binding pocket spanning both monomers in the functional dimeric unit, with the aminocoumarin moiety of SD8 buried in the protein core, while the angucyclic polyketide moiety is partially exposed to bulk solvent. Through comparisons of the structures, we postulate a derepression mechanism for SimR that invokes rigid-body motions of the subunits relative to one another, coupled with a putative locking mechanism to restrict further conformational change.

Improved Mutasynthetic Approaches for the Production of Modified Aminocoumarin Antibiotics

This study reports improved mutasynthetic approaches for the production of aminocoumarin antibiotics. Previously, the mutasynthetic production of aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1H-NMR spectroscopy.

Overexpression, purification and characterization of SimL, an amide synthetase involved in simocyclinone biosynthesis

Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tu 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4'-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.

Simocyclinone D8, an Inhibitor of DNA Gyrase with a Novel Mode of Action

We have characterized the interaction of a new class of antibiotics, simocyclinones, with bacterial DNA gyrase. Even though their structures include an aminocoumarin moiety, a key feature of novobiocin, coumermycin A(1), and clorobiocin, which also target gyrase, simocyclinones behave strikingly differently from these compounds. Simocyclinone D8 is a potent inhibitor of gyrase supercoiling, with a 50% inhibitory concentration lower than that of novobiocin. However, it does not competitively inhibit the DNA-independent ATPase reaction of GyrB, which is characteristic of other aminocoumarins. Simocyclinone D8 also inhibits DNA relaxation by gyrase but does not stimulate cleavage complex formation, unlike quinolones, the other major class of gyrase inhibitors; instead, it abrogates both Ca(2+)- and quinolone-induced cleavage complex formation. Binding studies suggest that simocyclinone D8 interacts with the N-terminal domain of GyrA. Taken together, our results demonstrate that simocyclinones inhibit an early step of the gyrase catalytic cycle by preventing binding of the enzyme to DNA. This is a novel mechanism for a gyrase inhibitor and presents new possibilities for antibacterial drug development.

Production of 8′-Halogenated and 8′-Unsubstituted Novobiocin Derivatives in Genetically Engineered Streptomyces coelicolor Strains

In the present study, we produced a hybrid antibiotic, carrying a chlorine atom instead of a methyl group at position 8 of the aminocoumarin moiety of novobiocin. This compound was not accessible by conventional gene inactivation/gene expression experiments due to difficulties in the genetic manipulation of the novobiocin producer Streptomyces spheroides. However, the desired compound was obtained after modification of the novobiocin biosynthetic gene cluster by using lambda-Red-mediated recombination in Escherichia coli, followed by integration of the resulting modified cosmid into the phiC31 attachment site of Streptomyces coelicolor and coexpression of the halogenase Clo-hal of clorobiocin biosynthesis. The halogenase BhaA, responsible for chlorination of tyrosyl moieties of the glycopeptide antibiotic balhimycin, was unable to functionally replace the halogenase Clo-hal, suggesting that the two enzymes have different substrate specificities.

An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489.

The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A1, i.e. between the central 3-methylpyrrole-2,4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mn2+ or Mg2+. The apparent Km values were determined as 26 micro m for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 micro m for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference from the genuine acyl substrate of CouL.

Cloning and analysis of the simocyclinone biosynthetic gene cluster of Streptomyces antibioticus Tü 6040.

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.

Genetic and functional analysis of novobiocin non‐producing mutants of Streptomyces niveus

Analysis of a series of 10 novobiocin non‐producing (Nov−) mutants of Streptomyces niveus failed to detect the production of novobiocin in all pairwise combinations in cosynthesis tests. Novobiocin production was restored in nine mutants when the growth medium was supplemented with the pathway intermediates, novobiocic acid and descarbamylnovobiocin, indicating that the mutants are blocked in the synthesis of the amino coumarin moiety (Ring B). Thin layer chromatographic analysis confirmed this by showing that each mutant was able to synthesize the benzoic acid component (Ring A) but not novobiocic acid (the fusion product of Rings A and B) or descarbamylnovobiocin. A study of the novobiocin resistance characteristics of the 10 mutants showed resistance levels considerably lower than for wild type. The Nov− mutants could be classified into four phenotypic groupings (Ib+ Is+, Ib+ Is−, Ib− Is+ and Ib− Is−) on the basis of their ability to be induced to high level resistance (Ib) by an inducing factor produced by wild‐type mycelium and their ability to induce high level resistance (Is) in wild type.