Substrate binding and cleavage mechanism of rubber oxygenase RoxA

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The aim of this proposal is to determine the biochemical cleavage mechanism of natural rubber (polyisoprene) by rubber oxygenase RoxA. This will be done by (i) identification of catalytically important residues of the active site of RoxA that participate in dioxygen binding, (ii) by identification of residues that are responsible for specific binding of the polymeric (hydrophobic) substrate to RoxA and by (iii) determination of changes of the RoxA molecule upon binding of substrate, substrate analogs or inhibitors (UVvis spectroscopy, EPR signals, product pattern, 3D structure). We will construct, purify and biochemically and biophysically characterize a set of RoxA muteins and will determine the structures of RoxA and RoxA Phe317Tyr mutein after co-crystallization with self-prepared substrate analogs. Together with our already funded project on the function of latex clearing protein (Lcp) we want to compare the biochemical cleavage mechanisms of RoxA and Lcp in order to understand how nature developed two different solutions for the same biochemical task, namely the oxidative cleavage of natural rubber by RoxA and Lcp.