Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum

Thiolase is the first enzyme catalysing the condensation of two acetyl-coenzyme A (CoA) molecules to form acetoacetyl-CoA in a dedicated pathway towards the biosynthesis of n -butanol, an important solvent and biofuel. Here we elucidate the crystal structure of Clostridium acetobutylicum thiolase ( Ca THL) in its reduced/oxidized states. Ca THL, unlike those from other aerobic bacteria such as Escherichia coli and Zoogloea ramegera , is regulated by the redox-switch modulation through reversible disulfide bond formation between two catalytic cysteine residues, Cys88 and Cys378. When Ca THL is overexpressed in wild-type C. acetobutylicum , butanol production is reduced due to the disturbance of acidogenic to solventogenic shift. The Ca THL V77Q/N153Y/A286K mutant, which is not able to form disulfide bonds, exhibits higher activity than wild-type Ca THL, and enhances butanol production upon overexpression. On the basis of these results, we suggest that Ca THL functions as a key enzyme in the regulation of the main metabolism of C. acetobutylicum through a redox-switch regulatory mechanism.