Über die Funktion und Struktur der Tyrosinase aus Streptomyces antibioticus

Für die Aufklärung der chemisch anspruchsvollen Monophenolase-Reaktion von Tyrosinasen wurde ein System entwickelt, um das Zielprotein aus dem Bakterium Streptomyces antibioticus in großen Mengen und mit hoher Reinheit zu isolieren. Zudem konnte ein hypothetischer Reaktionsmechanismus für die Monophenolase- und die Diphenolase-Aktivität der Tyrosinase formuliert werden. Die beiden Reaktionen der S. antibioticus-Tyrosinase wurden kinetisch analysiert und auf diesem Weg die Aktivität des Enzyms mit jener der sehr gut charakterisierten Tyrosinase aus dem Pilz Agaricus bisporus verglichen. Hierbei wurden signifikante Unterschiede festgestellt, die auf die verschiedenartigen Proteinstrukturen zurückgeführt wurden. Auch konnte gezeigt werden, dass einige sekundäre Pflanzenstoffe, die vor allem in Wein zu finden sind und in ihrer chemischen Struktur den Tyrosinasesubstraten ähnlich sind, die Aktivität dieses Enzyms maßgeblich beeinflussen. Das O2-Transportprotein Hämocyanin aus der Vogelspinne Eurypelma californicum, das wie die Tyrosinase zu der Familie der Typ-3-Kupferproteine gehört, ist nach chemischer Aktivierung zur Phenoloxidase zur enzymatischen Quervernetzung von Proteinen fähig. Die Tatsache, dass diese Quervernetzung auch das Hämocyanin selbst betrifft, sowie der erfolgreiche Nachweis von Hämocyanin in der Kutikula des genannten Organismus, legen die Vermutung nahe, dass die physiologische Funktion von Hämocyanin im Rahmen der Sklerotisierung des Exoskeletts in einer aktiven und passiven Beteiligung an Gerbungsprozessen im Integument besteht.

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.