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 Zn²⁺ ion coordinated by E8, H167, D291 and three water molecules. This is different from 2,6-DHBD from Rhizobium sporomusa, in which the Zn²⁺ 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.

Keywords: biocatalysis; dihydroxybenzoic acid decarboxylase; mutagenesis; structure elucidation; substrate recognition mechanism.