Dissecting the Mechanism of Biosynthesis of Sulfurated Biomolecules: The Case of tRNA Sulfuration

Sulfuration of biomolecules is a crucial biochemical process responsible for producing essential organic cofactors, coenzymes, vitamins, and other sulfurated biomolecules. In particular, thiomodified nucleosides in tRNA play a key role in ensuring the accuracy and efficiency of genetic translation. Although sulfurating enzymes have been studied structurally and biochemically since the early 2000s, detailed mechanistic analyses remain limited. In particular, the importance of reductants used in catalytic assays is often underappreciated.

In recent years, the use of anaerobic conditions has led to the discovery, in many of these enzymes, of an air-sensitive iron–sulfur cluster that is essential for catalysis. This led to the proposal of a catalytic mechanism involving a [4Fe–5S] intermediate, distinct from the previously accepted persulfide-based pathway. As this remains a debated topic, the current understanding of both mechanisms and the role of reductants warrants a critical evaluation.

The sulfur atom for the reaction is usually extracted from cysteine by a cysteine desulfurase (CD) and then used as the sulfur source by sulfurating enzymes, as noted by E throughout the article. Two main E classes are distinguished by their catalytic centers:

Overall, both E classes critically depend on two-electron reducing systems to sustain catalytic cycling. The presence or absence of reductants strongly influences enzyme activity and mechanisms, so that in vitro results, especially those using nonphysiological reductants like DTT, should be interpreted with caution.

Understanding the fundamental chemistry of Es is essential not only for elucidating their role in metabolism and cellular regulation but also for potential therapeutic targeting and biotechnological applications. A key remaining challenge is the identification of physiological reducing systems, a critical yet unresolved aspect of sulfuration biochemistry.