Fe-S clusters are ubiquitous cofactors found in all forms of life. Due to the toxicity of free iron and sulfide in the cells, Fe-S cluster assembly requires complex biosynthetic pathways. Fe-S cluster biosynthesis consists of three steps: (1) sulfur mobilization, (2) cluster assembly, and (3) cluster transfer. The sulfur mobilization step is conducted by pyridoxal-5’-phosphate (PLP)-dependent cysteine desulfurases (CSDs). The PLP cofactor binds a free cysteine substrate, abstracts a sulfur atom, and releases the resulting alanine byproduct. In the SUF pathway of Fe-S cluster biosynthesis, CSDs utilize shuttling proteins to transfer S to the Fe-S cluster scaffold complex. Although CSDs are primarily known for their function in S capture, previous studies have shown that CSDs are also active with selenocysteine as a substrate. Certain CSDs even display a higher activity with selenocysteine than cysteine, but it is unknown whether selenium can be transferred to shuttling proteins in the SUF pathway. In this work, we characterize the enzyme activities and kinetic parameters of Gram-positive CSDs with multiple substrates, and we determine whether Se transfer from CSDs to shuttling proteins in the SUF pathway can occur. The results of this work contribute to our understanding of how selenocysteine and Se may be used by proteins in Fe-S cluster biosynthesis pathways.

Activity analysis of bacterial cysteine desulfurases and their shuttling proteins with selenocysteine as the substrate

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ACS Fall 2022

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