1H NMR, Mechanism, and Mononuclear Oxidative Activity of the Antibiotic Metallopeptide Bacitracin: The Role of d-Glu-4, Interaction with Pyrophosphate Moiety, DNA Binding and Cleavage, and Bioactivity

摘要

The peptidyl antibiotic bacitracin (Bc) is one of the most widely used antibiotics which can bind divalent transition metal ions, including Mn(II), Co(II), Ni(II), Cu(II), and Zn(II). The metal binding is essential for its antimicrobial activity. Previous analysis of the hyperfine-shifted 1H NMR signals of Co(II)−Bc A1 revealed the structure of the metal binding environment and a potential hydrophobic site important for the bioactivity of this antibiotic. Co(II)−Bc in DMSO shows relatively sharper hyperfine-shifted 1H NMR signals compared with the spectrum acquired in an aqueous solution, allowing more thorough analysis of the signals with 1D and 2D NMR methods. Pyrophosphate and derivatives bind to Co(II)−Bc to form kinetically inert ternary complexes. The coordinated d-Glu-4 is found detached from the metal center of metallobacitracin upon trimetaphosphate binding, implying its role in the antibiotic activity of Bc. We further demonstrate in this report the structure−function relationship on desamido-Bc of low antibiotic activity by the use of NMR, wherein d-Glu-4 is suggested to be important for the bioactivity of Bc. The interaction of the phospho-moiety with Bc is also reflected by DNA binding, wherein metal-free Bc does not bind DNA, whereas various metal complexes of Bc do. Cu(II)−Bc was further demonstrated to bind and oxidatively cleave DNA under reduction conditions in the air. It also exhibited a significant oxidative activity toward catechol oxidation, showing enzyme-like saturation kinetics with kcat = 7.0 × 10−3 s−1 and kcat/Km = 2.1 M−1 s−1 aerobically and kcat = 0.38 s−1 and kcat/Km = 14.7 M−1 s−1 in the presence of 32 mM of H2O2. The binding of pyrophosphate moiety to metallobacitracin, the detachment of d-Glu-4, and the significant oxidative activity of Cu(II)−Bc provide further insights into the bioactivity of this metallopeptide and Cu−oxygen chemistry.