Comparative genomic insights into the action of suicide Thi4 thiazole synthases

Suicide Thi4 thiazole synthases are mononuclear metal enzymes that form the thiazole moiety of thiamin from NAD ⁺ , glycine, and a sulfur atom that is stripped from an active-site cysteine residue, causing enzyme inactivation. However, many prokaryotic Thi4 genes cluster on the chromosomal regions with genes encoding ThiS, ThiF, and other proteins that produce, relay, or use persulfide or thiocarboxylate sulfur. Such clusters include DUF6775 proteins, with domains of unknown function that feature a metal-binding motif. This comparative genomic evidence suggests that certain suicide Thi4s might use a persulfide or thiocarboxylate as sulfur donor instead of the active-site cysteine – i.e., that they can operate in a non-suicide mode – and that a metal cofactor reservoir supports Thi4 function. We explored both possibilities using Escherichia coli as a heterologous platform. A representative bacterial Thi4 (clustered with thiS and thiF ) complemented an E. coli Δ thiG (thiazole auxotroph) single mutant better than a Δ thiG Δ thiF Δ thiS triple mutant, indicating that this Thi4 interacts with the E. coli sulfide transfer chain. A representative archaeal DUF6775 protein was expressed in E. coli; however, the bound metal was not detected in protein prepared from inclusion bodies. Collectively, this evidence indicates that suicide Thi4s do not necessarily always operate suicidally with respect to the sulfur donor or autonomously with respect to the metal cofactor.