Mechanistic and structural insights into the itaconate-producing <i>trans</i>-aconitate decarboxylase Tad1

Itaconic acid belongs to the high-value precursors for the production of biomass-based industrial compounds. It originates from the tricarboxylic acid cycle, and depending on the organism, it is produced by different biosynthetic routes. The basidiomycete fungus <i>Ustilago maydis</i> synthesizes itaconic acid via isomerization of <i>cis</i>-aconitic acid to <i>trans</i>-aconitic acid, and subsequent decarboxylation catalyzed by the <i>trans</i>-aconitate decarboxylase Tad1, which belongs to the aspartase/fumarase superfamily. Since no other decarboxylase has been identified within this protein superfamily, Tad1 constitutes a novel type of decarboxylase. Here, we present high-resolution crystal structures of Tad1, which, together with mutational analysis and nuclear magnetic resonance spectroscopy measurements, provide insight into the molecular mechanism of Tad1-dependent decarboxylation. Specifically, our study shows that decarboxylation is favored in acidic conditions, requires protonation as well as migration of a double bond, and coincides with structural rearrangements in the catalytic center. In summary, our study elucidates the molecular mechanism underlying a novel type of enzymatic decarboxylation and provides a starting point for protein engineering aimed at optimizing the efficient production of itaconic acid.