Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized -- but only from archaeal methanogens that are anaerobic, O-2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli Delta thiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O-2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared with the structures of THI4s from O-2-sensitive methanogens, but is consistent with an alternative catal metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

Automated summary

Plant and fungal THI4 thiazole synthases produce thiamin thiazole in aerobic conditions via a single-turnover suicide reaction, while multiple-turnover THI4s can operate in aerobic, mild, low-sulfide conditions. These THI4s lacking active-site Cys have been found in both anaerobic archaeal methanogens and aerobic mesophiles, indicating a wider potential range of operation for THI4 enzymes.