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

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Plant and fungal THI4 enzymes synthesize the thiamin thiazole moiety aerobically via a single-turnover reaction.
• Multiple-turnover (catalytic) THI4s, typically found in anaerobic archaea, use sulfide as a sulfur donor and prefer iron as a cofactor.
• The operability of catalytic THI4s in aerobic, low-sulfide environments was previously unknown.

Purpose of the Study:

• To investigate the potential for multiple-turnover THI4 enzymes to function in aerobic conditions.
• To identify the sulfur donor and metal cofactor utilized by aerotolerant non-Cys THI4s.
• To elucidate the structural basis for aerotolerance in THI4 enzymes.

Main Methods:

• Genomic survey to identify non-Cys THI4s in aerobic bacteria.
• Functional complementation assays using an Escherichia coli thiazole auxotroph under aerobic conditions.
• Determination of the crystal structure of Thermovibrio ammonificans THI4.

Main Results:

• Sixteen of 23 tested non-Cys THI4s demonstrated catalytic activity in aerobic conditions.
• Enzyme activity was enhanced by supplying cysteine, indicating the use of sulfide or a metabolite as the sulfur donor.
• Structural and complementation data suggest cobalt, rather than iron, as the preferred metal cofactor for aerotolerant THI4s.

Conclusions:

• Catalytic THI4 enzymes can operate effectively in aerobic environments.
• The metal cofactor, likely cobalt, plays a crucial role in determining the aerotolerance of THI4 enzymes.
• This work broadens the understanding of thiamin biosynthesis pathways in diverse microbial habitats.