| Xanthobacter strain Py2 is one of several bacteria capable of growth using aliphatic alkenes and epoxides as carbon and energy sources. The oxidation of alkenes is initiated by an inducible alkene monooxygenase which inserts an oxygen atom derived from molecular oxygen into the olefin bond, producing the corresponding epoxide. The epoxides thus formed are further metabolized enzymatically by a pathway that had not been studied at the outset of the research described in this dissertation. These studies led to the elucidation of a new microbial pathway of aliphatic epoxide metabolism that involves epoxide ring opening and carboxylation, forming a beta-keto acid as product. This reaction is catalyzed by an enzyme designated "epoxide carboxylase." In the course of these studies a second epoxide conversion, isomerization to a ketone, was characterized as a fortuitous reaction catalyzed by epoxide carboxylase in the absence of CO2. Thus, these studies have identified two previously unknown biological epoxide transformations; After elucidating the pathway of epoxide metabolism, the purification and characterization of alkene monooxygenase was initiated. Alkene monooxygenase was found to be a multiprotein enzyme complex consisting of (1) the epoxygenase, which contains the active site for olefin epoxidation, (2) a reductase, which oxidizes NADH, providing the reducing equivalents required for alkene oxidation and oxygen reduction, (3) a rieske-type ferredoxin, which accepts electrons from the reductase, and transfers them to the epoxygenase, and (4) a small effector protein required for steady-state alkene epoxidation. Each of the four alkene monooxygenase components was purified to homogeneity and biochemically and spectroscopically characterized. This work provided the first identification of a four-component monooxygenase utilizing a two-component electron transfer scheme for the activation and oxidation of an aliphatic hydrocarbon. |
