Macrophomate synthase which catalyzes unusual multiple reactions from 2-pyrone (3) to macrophomic acid (1) has been purified in a homogenous state. The macrophomate synthase is a dimeric enzyme which requires Mg2+ as a co-factor and whose molecular mass is 40 kDa. Study on substrate specificity showed that the enzyme is capable of converting various 2-pyrones to the corresponding benzoates.