Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase.

Selenium is a vital micronutrient in many organisms. While traces are required for microbial utilization, excess amounts are toxic; thus, selenium can be regarded as a biological double-edged sword. Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. We have identified SclA, a NifS-like protein in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for l-selenocysteine over l-cysteine. It is known that Asp-146 is required for selenocysteine specificity in the human selenocysteine lyase. Thus, using computational biology, we compared the bacterial and mammalian enzymes and identified His-100, an Asp-146 ortholog in SclA, and generated site-directed mutants in order to study the residue's potential role in the l-selenocysteine discrimination mechanism. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying Michaelis-Menten behavior towards l-selenocysteine, but His-100 was not found to be essential for this activity. Additionally, l-cysteine acted as a competitive inhibitor of all enzymes with higher affinity than l-selenocysteine. Finally, we discovered that SclA exhibited low activity with l-cysteine as a poor substrate regardless of mutations. We conclude that His-100 is not required for l-selenocysteine specificity, underscoring the inherent differences in discriminatory mechanisms between bacterial NifS-like proteins and mammalian selenocysteine lyases.