2,3-Dihydroxybenzoic Acid Decarboxylase from Fusarium oxysporum: Crystal Structures and Substrate Recognition Mechanism |
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Authors: | Mengkun Song Xuemei Zhang Dr. Weidong Liu Dr. Jinghui Feng Yunfeng Cui Dr. Peiyuan Yao Prof. Dr. Min Wang Prof. Dr. Rey-Ting Guo Prof. Dr. Qiaqing Wu Prof. Dr. Dunming Zhu |
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Affiliation: | 1. Key Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science and Technology, Tianjin, 300457 P. R. China National Engineering Laboratory for Industrial Enzymes, Tianjin Engineering Research Center of Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China;2. National Engineering Laboratory for Industrial Enzymes, Tianjin Engineering Research Center of Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China;3. Key Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science and Technology, Tianjin, 300457 P. R. China |
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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 R233Δ 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. |
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Keywords: | biocatalysis dihydroxybenzoic acid decarboxylase mutagenesis structure elucidation substrate recognition mechanism |
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