Abstract 1753 Investigating hydroalkylases as biocatalysts

Micro-organisms can thrive in seemingly harsh environments, including anoxic (i.e. oxygen-free), crude-oil polluted environments. Many of these microbes can not only survive in crude oil but can also use various hydrocarbons within crude oil as carbon sources. Adding functional handles to hydrocarbons and thereby activating them for further metabolism without O2 is a challenging task, as their C–H bonds are relatively inert. Anoxic hydrocarbon activation is accomplished by enzymes that convert C–H bonds to C–C or C–O bonds though different mechanisms (e.g. olefin hydroalkylation, carboxylation, hydroxylation using water). The predominant activation method is an olefin hydroalkylation mechanism whereby a hydrocarbon C–H bond is homolytically cleaved, the hydrocarbyl radical is added to one side of the alkene, and the hydrogen atom is added to the other side of the alkene. This type of reaction is a synthetically attractive method for forming C–C bonds, as it can set up to three contiguous stereocenters without byproducts or pre-functionalized substrates. This chemistry is enabled by a class within the large glycyl radical enzyme superfamily known as X-succinate synthases (XSSs). Elegant structural and biochemical studies have shed light on how XSSs are able to catalyze this reaction using both aromatic and saturated hydrocarbons; however, an inability to catalyze hydoalkylations using purified enzymes hampered efforts to fully characterize these enzymes. Recently, we have developed a method to catalyze these reactions in vitro. Using this in vitro platform, we are now able to determine kinetics for XSSs, understand the function of accessory subunits, explore their substrate scope, and screen variant libraries for altered substrate scope. These results will set the stage to both better understand the mechanism of these fascinating catalysts and explore their use as synthetically useful, selective C–H functionalization biocatalysts. This work was funded by an NIH MOSAIC R00 (GM145910) and Michigan State University.