Abstract
Anaerobic choline deamination catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as a major route for trimethylamine (TMA) production within anaerobic environments, including the human gut. The association of this microbial metabolite and its downstream products with diseases such as atherosclerosis and chronic kidney disease has driven the need for a better molecular understanding of TMA-generating enzymes. Our previous work has shown that generating the critical, glycine-centered radical species on CutC requires posttranslational modification by an S-adenosyl-l-methionine (SAM)-dependent radical-activating protein (CutD) harboring an oxygen-sensitive [4Fe-4S] cofactor. In this chapter, we describe our strategy to heterologously express and purify Desulfovibrio alaskensis G20 CutD in Escherichia coli and reconstitute its iron-sulfur center under anaerobic conditions. In addition, we present the methods we have developed to characterize the activity of CutD and utilize this enzyme in conjunction with purified CutC to gain an unprecedented insight into the anaerobic C-N cleavage of choline.
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