ABSTRACT
Showdomycin is a C-nucleoside bearing an electrophilic maleimide base. Herein, the biosynthetic pathway of showdomycin is presented. The initial stages of the pathway involve non-ribosomal peptide synthetase (NRPS) mediated assembly of a 2-amino-1H-pyrrole-5-carboxylic acid intermediate. This intermediate is prone to air oxidation whereupon it undergoes oxidative decarboxylation to yield an imine of maleimide, which in turn yields the maleimide upon acidification. It is also shown that this pyrrole intermediate serves as the substrate for the C-glycosidase SdmA in the pathway. After coupling with ribose 5-phosphate, the resulting C-nucleoside undergoes a similar sequence of oxidation, decarboxylation and deamination to afford showdomcyin after exposure to air. These results suggest that showdomycin could be an artifact due to aerobic isolation; however, the autoxidation may also serve to convert an otherwise inert product of the biosynthetic pathway to an electrophilic C-nucleotide thereby endowing showdomycin with its observed bioactivities.
Subject(s)
Glycosides/metabolism , Peptide Synthases/metabolism , Pyrroles/metabolism , Showdomycin/biosynthesis , Glycosides/chemistry , Molecular Conformation , Oxidation-Reduction , Peptide Synthases/chemistry , Pyrroles/chemistry , Showdomycin/chemistryABSTRACT
Showdomycin is a potent nucleoside antibiotic that displays a high structural similarity to uridine and pseudouridine. No detailed target analysis of this very unusual electrophilic natural product has been carried out so far. To unravel its biological function, we synthesized a showdomycin probe that can be appended with a fluorophor or a biotin marker via click chemistry and identified diverse enzymes which were important for either the viability or virulence of pathogenic bacteria. Our results indicate that the antibiotic effect of showdomycin against Staphylococcus aureus may be due to the inhibition of various essential enzymes, especially MurA1 and MurA2, which are required for cell wall biosynthesis. Although real-time polymerase chain reaction revealed that the MurA2 gene was expressed equally in four S. aureus strains, our probe studies showed that MurA2 was activated in only one multiresistant S. aureus strain, and only this strain was resistant to elevated concentrations of the MurA inhibitor fosfomycin, suggesting its potential role as an antibiotic bypass mechanism in the case of MurA1 inhibition. Moreover, we utilized this tool to compare enzyme profiles of different pathogenic strains, which provided unique insights in regulatory differences as well as strain-specific signatures.
Subject(s)
Anti-Bacterial Agents/chemistry , Bacteria/enzymology , Enzymes/analysis , Showdomycin/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacteria/pathogenicity , Binding Sites , Biological Products/chemistry , Biological Products/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzymes/metabolism , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Showdomycin/pharmacologyABSTRACT
Showdomycin is a naturally maleimide antitumor antibiotic of the C-nucleoside, it inhibits the nucleic acid synthesis in bacteria. Conformational analysis and geometry optimization of showdomycin was performed according to the Hartree-Fock (HF) calculation method by ArgusLab 4.0.1 software. The minimum potential energy is calculated by geometry convergence function by ArgusLab software. The most feasible position for the drug to interact with the receptor was found to be 0.269696 K.cal/mole.
Subject(s)
Antibiotics, Antineoplastic/chemistry , Computer Simulation , Models, Molecular , Showdomycin/chemistry , Software , Molecular Conformation , Quantitative Structure-Activity RelationshipABSTRACT
A new, convenient, and short synthesis of 2'-deoxyshowdomycin, along with an improved procedure for the preparation of showdomycin, have been presented. A single-crystal X-ray structure of 1-benzyl-2'-deoxyshowdomycin (9) has been reported. Conformational studies using C.D. indicated that showdomycin exists predominantly in an anti conformation in aqueous solution. Molecular mechanics calculations using AMBER point to comparable binding energy of showdomycin-adenosine pair with the natural uridine-adenosine pair, but with a significant base-ribose conformational deviation from the natural array in the former. Implications of such a conformational deviation on tumor and viral replications have been discussed. Base-pairing studies employing high resolution NMR spectroscopy indicate that both showdomycin and epishowdomycin base-pair with adenosine-5'-monophosphate (AMP); however, while showdomycin also shows evidence of stacking, that was absent in epishowdomycin. Molecular modeling studies using QUANTA/CHARMm show that showdomycin is capable of forming a homopolymer duplex by base-pairing with poly(A), but with a considerably broader and deeper major groove. A heteropolymer duplex with a single insert of showdomycin exhibits tighter coiling at the point of insertion. A ten-picosecond dynamics simulation of the above heteroduplex revealed relaxation of the helix with disruption of H-bonding for two base pairs on either side of the insertion point, forming a large central cavity.
