Characterization of an S-adenosylmethionine Dependent X-Succinate Synthase Activating Enzyme.

Hydrocarbons and, unfortunately, hydrocarbon waste are ubiquitous in natural and man-made environments. It is unsurprising, then, that many microbes are capable of metabolizing hydrocarbons for use as carbon sources. Such microbes can be responsible for biodeterioration, leading to environmental damage, but paradoxically also play a role in bioremediation. As a result, an understanding of their mechanisms of action, and the ability to reproduce those mechanisms in vitro, is highly sought-after. One such hydrocarbon degradation mechanism kicks off with the anaerobic addition of hydrocarbons to fumarate by a class of enzymes known as X-succinate synthases (XSSs). These enzymes use a glycyl radical cofactor to accomplish this difficult chemistry under mild conditions. This cofactor must be installed by a radical S-adenosylmethionine (SAM) activating enzyme, which has presented a hurdle to obtaining activity in vitro. The most well-characterized XSS, known as benzylsuccinate synthase (BSS), adds toluene to fumarate to form benzylsuccinate. Unfortunately, its corresponding activating enzyme (AE) does not solubly express in E. coli, which has limited research into the mechanism of XSS activation. To circumvent this barrier, we took a genome-mining approach to identify homologs of BSS-AE, yielding soluble homologs that expressed well in E. coli. Using EPR spectroscopy and LC-MS/MS, we have demonstrated that the 4Fe4S cluster within one of these AEs is able to be reduced and that this AE is able to cleave SAM. Moreover, we have expressed this particular homolog's corresponding XSS and found that hydroalkylation activity can be reconstituted in vitro, as observed by LC-MS/MS. It is our hope that this system will allow for a greater understanding of XSS-AEs and their corresponding XSSs, as well as for the development of these enzymes as synthetically useful catalysts.