2,3‐Dihydroxybenzoic Acid Decarboxylase from Fusarium oxysporum: Crystal Structures and Substrate Recognition Mechanism

Abstract A 2,3‐dihydroxybenzoic acid decarboxylase from Fusarium oxysporum (2,3‐DHBD_Fo) has a relatively high catalytic efficiency for the decarboxylation of 2,3‐dihydroxybenzoic acid (DHBA) and carboxylation of catechol, thus it has a different substrate spectrum from other benzoic acid decarboxylases. We have determined the structures of 2,3‐DHBD_Fo in its apo form and complexes with catechol or 2,5‐dihydroxybenzoic acid at 1.55, 1.97, and 2.45 Å resolution, respectively. The crystal structures of 2,3‐DHBD_Fo show that the enzyme exists as a homotetramer, and each active center has a Zn 2+ ion coordinated by E8, H167, D291 and three water molecules. This is different from 2,6‐DHBD from Rhizobium sporomusa , in which the Zn 2+ ion is also coordinated with H10. Surprisingly, mutation of A10 of 2,3‐DHBD_Fo to His resulted in almost complete loss of the enzyme activity. Enzyme‐substrate docking and site‐directed mutation studies indicate that residue R233 Δ interacts with the 3‐hydroxy group of 2,3‐DHBA, and plays an important role in substrate recognition for this enzyme, thus revealing the molecular basis 2,3‐dihydroxybenzoic acid decarboxylase.