The Catalytic Promiscuity of TSTD1-Like Sulfurtransferases Originates from a Bifaceted Active Site

The development of original synthesis methodologies using eco-friendly biocatalytic strategies is growing exponentially. One of the limiting factors remains the availability of efficient biocatalysts, particularly for the formation of C–S bonds. Thiosulfate sulfurtransferases (TSTs) are promising candidates that catalyze sulfur transfer from thiosulfate (TS) through the formation of the persulfide intermediate on the catalytic cysteine. However, the mechanism by which sulfur–sulfur bond cleavage occurs remains to be elucidated. To address this gap of knowledge, the mechanism of persulfide formation was investigated on human TSTD1 as a TST model through structure/function relationships studies. We showed that the persulfide formation by sulfur–sulfur bond cleavage of TS is critically dependent on negative charge neutralization and that the sulfur transfer process is “proton-independent”. Surprisingly, TSTD1 is more efficient in catalyzing carbon–sulfur than sulfur–sulfur bond cleavage, with the former including a water-mediated protonation for which Q80 is essential. This catalytic promiscuity mainly originates from two paired residues located on opposite faces within the active site and specifically dedicated to sulfur–sulfur bond cleavage for the E31/R84 pair and carbon–sulfur bond cleavage for the Q80/R108 pair, respectively. However, we showed for other TST members that it can also be achieved through alternative catalytic strategies.