Crystal structures and substrate recognition mechanism of 2, 3-dihydroxybenzoic acid decarboxylase from Fusarium oxysporum.

: A 2,3-dihydroxybenzoic acid decarboxylase from Fusarium oxysporum (2,3-DHBD_Fo) has relatively high catalytic efficiency for decarboxylation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and carboxylation of catechol, thus has a different substrate spectrum from other benzoic acid decarboxylases. We have determined the structures of 2,3-DHBD_Fo in apo form, complexes with catechol or 2,5-dihydroxybenzoic acid (2,5-DHBA) at 1.55 A,1.97 A and 2.45 A resolution, respectively. The crystal structures of 2,3-DHBD_Fo show that the enzyme exists as 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 Zn 2+ ion is also coordinated with H10. Surprisingly, mutation of A10 of 2,3-DHBD_Fo to H almost resulted in loss of the enzyme activity. The enzyme-substrate docking and site-directed mutation studies indicate that residue R233 △ interacts with the 3-hydroxyl group of substrate 2,3-DHBA, and plays an important role for the substrate recognition of this enzyme, revealing the molecular basis of being a 2,3-dihydroxybenzoic acid decarboxylase.