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Fungal peroxygenases represent an exciting new enzyme class for stereo-selective hydroxylation reactions. They are capable of the oxyfunctionalisation of a large, diverse scope of substrates including alkanes and steroids as well as the heteroatoms sulfur and nitrogen. The outstanding activities and stabilities as well as their reliance on hydrogen peroxide as co-substrate renders it a highly interesting biocatalyst.
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Background/Aim: Tubugi-1 is a more stable and accessible synthetic counterpart of natural tubulysins. This study aimed to evaluate its cytotoxic potential against anaplastic human melanoma cells. Materials and Methods: The viability of A-375 cells was determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assay. The type of cell death and proliferative rate were investigated using flow cytometry and fluorescent microscopy, while the molecular background was evaluated by western blot. Results: Tubugi-1 reduced the viability of A-375 cells, inducing massive micronucleation, followed by augmented expression of inhibitor of nuclear factor-κB and caspase-2, typical of a mitotic catastrophe. Disturbed proliferation and G2M block with prominent caspase activity, weakened the expression of B-cell lymphoma 2 and B-cell lymphoma 2-associated X transient up-regulation, coexisted with intensive autophagy. Specific inhibition of autophagy by chloroquine resulted in conversion from mitotic catastrophe to rapid apoptosis. Conclusion: Multilevel anticancer action of tubugi-1 is extended by co-application of an autophagy inhibitor, giving a new dimension in further preclinical advancement of this potential agent.
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The screening effort of large protein variant libraries renders the probability of coincidental discovering a new enzyme with non-natural activity to almost zero - the so-called numbers problem. Insights into the origin of life, evolution and enzymatic promiscuity, combined with the inspiration of methods from organic chemistry, offer solutions for this problem. With the newly discovered enzymes synthetic micro production units shall be established in a Leibniz Research Cluster where engineering and biotechnology are combined.
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Late stage enzymatic prenylation and methylation are means to diversify (natural) compounds and to specify their functions. In eukaryotes and microbes, these steps are performed by large enzyme families, the prenyl and methyl transferases, which modify various types of small molecules, like isoprenoids, phenolics or alkaloids, but also DNA and proteins. We investigate the theoretical basis of these processes and possible commercial applications in synthetic chemistry.
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In Arabidopsis, deactivation of cyclin-dependent kinases via phosphorylation has no function in cell proliferation, growth, and stress response. In other eukaryotes, however, this is mandatorily required for maintaining genomic integrity.