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bioRxiv · 10.1101/2020.02.07.939272

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

Johnstone, M. A.·Nelson, S. J.·Groesbeck, C. V.·Self, W. T.
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Abstract

Selenium is a vital micronutrient in many organisms. While traces are required for utilization by the microbe, 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 selenocysteine lyase in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for L-selenocysteine over L-cysteine. It is known that a single residue in the human selenocysteine lyase, D146, is considered to control selenocysteine specificity. Thus, using computational biology, we identified H100, a D146 ortholog in SclA, and generated mutant enzymes with site-directed mutagenesis. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying levels of activity towards L-selenocysteine, suggesting a catalytic role for H100. Additionally, L-cysteine acted as a competitive inhibitor towards all enzymes with higher affinity than L-selenocysteine. Our findings offer key insight into the molecular mechanisms behind selenium-over-sulfur specificity and may further elucidate the role of selenocysteine lyases in vivo.

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Johnstone, M. A., Nelson, S. J., Groesbeck, C. V., Self, W. T.. 2020-02-09. Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase. https://doi.org/10.1101/2020.02.07.939272

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