Rapid generation of hydrogen peroxide contributes to the complex cell death induction by the angucycline antibiotic landomycin E.

Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH• effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main cardiotoxic mechanism of Dx, LE might represent a better tolerable drug candidate for further (pre)clinical development.

Molecular insight into substrate recognition and catalysis of Baeyer-Villiger monooxygenase MtmOIV, the key frame-modifying enzyme in the biosynthesis of anticancer agent mithramycin.

Baeyer-Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C-C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed us to probe the substrate binding pocket of MtmOIV and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.

Baeyer-Villiger C-C bond cleavage reaction in gilvocarcin and jadomycin biosynthesis.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical of the gilvocarcin-type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxyoxepinone intermediate, leading to important conclusions regarding the cleavage mechanism.

Roles of the synergistic reductive O-methyltransferase GilM and of O-methyltransferase GilMT in the gilvocarcin biosynthetic pathway.

Two enzymes of the gilvocarcin biosynthetic pathway, GilMT and GilM, with unclear functions were investigated by in vitro studies using purified, recombinant enzymes along with synthetically prepared intermediates. The studies revealed GilMT as a typical S-adenosylmethionine (SAM) dependent O-methyltransferase, but GilM was identified as a pivotal enzyme in the pathway that exhibits dual functionality in that it catalyzes a reduction of a quinone intermediate to a hydroquinone, which goes hand-in-hand with a stabilizing O-methylation and a hemiacetal formation. GilM mediates its reductive catalysis through the aid of GilR that provides FADH(2) for the GilM reaction, through which FAD is regenerated for the next catalytic cycle. This unusual synergy eventually completes the biosynthesis of the polyketide-derived defuco-gilvocarcin chromphore.