Towards the Enzymatic Mineralization of Siloxanes

Through the synthesis of novel chemical entities, the global chemical industry has contributed significantly to the destabilization of the Earth system. Chapter I outlines the magnitude of this environmental problem and discusses the upsides and drawbacks of three mechanisms used to address it: physicochemical approaches, microbial degradation, and enzymatic bioremediation, specifically highlighting the protein engineering technique of directed evolution in support of the latter. Chapter II then provides an example emblematic of the power directed evolution has by degrading a specific class novel of chemical entities, volatile methylsiloxanes (VMS). VMS are anthropogenic, non-biodegradable chemicals produced at megaton-per-year scale and considered emerging environmental contaminants. We report on the discovery, and subsequent engineering, of cytochrome P450BM3 variants which break silicon–carbon bonds in linear and cyclic VMS, as well as propose a mechanism by which these enzymes catalyze the first known biological Si–C bond cleavage. VMS could not now be considered “biodegradable,” however, as biodegradation is more appropriately understood as “mineralization,” reducing a compound solely to carbon dioxide, water, and/or inorganics; mineralization is critical because breakdown products of the parent compound may be just as, if not more, problematic than the parent which, for VMS, means their monomeric subunit, dimethylsilanediol (DMSD). Chapter III addresses this shortcoming by describing the discovery of a wild-type enzyme, the sulfite reductase CysI, which can cleave both Si–C bonds of DMSD, representing the capacity for its mineralization. Given the curious nature of a reductase performing oxidative chemistry, the mechanism of CysI was also probed, finding that it has latent NADPH oxidase and peroxygenase activity. Overall, this thesis illustrates how enzymes, and the new-to-nature chemistries imparted by directed evolution, may be the most suitable mechanism for the degradation of novel chemical entities, even potentially mineralizing them.