ABSTRACT
Synthetic chain terminators were used to capture the biosynthetic intermediates from a partially reducing iterative type I polyketide synthase, which is integrated into a multimodular biosynthesis enzyme. The off-loaded metabolites clarified the timing of ketoreduction and aromatization in the assembly of the antibiotic micacocidin.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Organometallic Compounds/metabolism , Polyketide Synthases/metabolism , Ralstonia solanacearum/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Biosynthetic Pathways , Organometallic Compounds/chemistry , Oxidation-Reduction , Polyketide Synthases/chemistry , Protein Structure, Tertiary , Ralstonia solanacearum/chemistry , Substrate SpecificityABSTRACT
Micacocidin is a promising natural product for the treatment of Mycoplasma pneumoniae infections. In the biosynthesis of this antibiotic, a fatty acid-AMP ligase (FAAL) activates the starter unit hexanoic acid as acyl-adenylate and forwards it to an iteratively acting polyketide synthase. Biochemical analysis of the FAAL revealed an extended substrate tolerance, thereby opening the door for the modification of a micacocidin residue that is barely accessible via semisynthesis. A total of six new analogues were generated by precursor-directed biosynthesis in this study and profiled against M. pneumoniae.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biological Products/pharmacology , Mycoplasma pneumoniae/drug effects , Organometallic Compounds/pharmacology , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Biological Products/chemistry , Biological Products/metabolism , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Structure , Organometallic Compounds/chemistry , Organometallic Compounds/metabolism , Structure-Activity RelationshipABSTRACT
Micacocidin is a thiazoline-containing natural product from the bacterium Ralstonia solanacearum that shows significant activity against Mycoplasma pneumoniae. The presence of a pentylphenol moiety distinguishes micacocidin from the structurally related siderophore yersiniabactin, and this residue also contributes to the potent antimycoplasma effects. The biosynthesis of the pentylphenol moiety, as deduced from bioinformatic analysis and stable isotope feeding experiments, involves an iterative type I polyketide synthase (iPKS), which generates a linear tetraketide intermediate from acyl carrier protein-tethered hexanoic acid by three consecutive, decarboxylative Claisen condensations with malonyl-coenzyme A. The final conversion into 6-pentylsalicylic acid depends on a ketoreductase domain within the iPKS, as demonstrated by heterologous expression in E. coli and subsequent site-directed mutagenesis experiments. Our results unveil the early steps in micacocidin biosynthesis and illuminate a bacterial enzyme that functionally resembles fungal polyketide synthases.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Organometallic Compounds/metabolism , Polyketide Synthases/metabolism , Acyl Carrier Protein/chemistry , Acyl Carrier Protein/genetics , Acyl Carrier Protein/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Escherichia coli/metabolism , Isotope Labeling , Malonyl Coenzyme A/metabolism , Mutagenesis, Site-Directed , Mycoplasma pneumoniae/drug effects , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Polyketide Synthases/genetics , Ralstonia solanacearum/chemistryABSTRACT
A genome mining study led to the identification of a previously unrecognised siderophore biosynthesis gene cluster in the nitrogen-fixing bacterium Cupriavidus taiwanensis LMG19424. Based upon predicted structural residues, a convenient strategy for an NMR-assisted isolation of the associated metabolite was designed. The structure of the purified siderophore, taiwachelin, was fully characterized by spectroscopic methods and chemical derivatisation.
Subject(s)
Cupriavidus/chemistry , Cupriavidus/genetics , Genomics , Lipopeptides/chemistry , Siderophores/chemistry , Computational Biology , Magnetic Resonance Spectroscopy , Spectroscopy, Fourier Transform InfraredABSTRACT
The bacterium Cupriavidus necator H16 produces a family of linear lipopeptides when grown under low iron conditions. The structural composition of these molecules, exemplified by the main metabolite cupriachelin, is reminiscent of siderophores that are excreted by marine bacteria. Comparable to marine siderophores, the ferric form of cupriachelin exhibits photoreactive properties. Exposure to UV light induces an oxidation of its peptidic backbone and a concomitant reduction of the coordinated Fe(III). Here, we report the genomics-inspired isolation and structural characterization of cupriachelin as well as its encoding gene cluster, which was identified by insertional mutagenesis. Based upon the functional characterization of adenylation domain specificity, a model for cupriachelin biosynthesis is proposed.
Subject(s)
Cupriavidus necator/chemistry , Cupriavidus necator/metabolism , Siderophores/biosynthesis , Siderophores/chemistry , Molecular Structure , Oxidation-Reduction , Ultraviolet RaysABSTRACT
A genome mining study in the plant pathogenic bacterium Ralstonia solanacearum GMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated in R. solanacearum GMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes of R. solanacearum.