Laboratory for Conservation and Utilization of Bio-Resources, Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China.
PLoS One. 2011 Jan 21;6(1):e16262. doi: 10.1371/journal.pone.0016262.
Microbial ferulic acid decarboxylase (FADase) catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD) superfamily. Structural analysis revealed that FADase catalyzed reactions by an "open-closed" mechanism involving a pocket of 8 × 8 × 15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer.
微生物阿魏酸脱羧酶（FADase）通过非氧化脱羧作用将阿魏酸转化为 4-羟基-3-甲氧基苯乙烯（4-乙烯基愈创木酚）。在这里，我们报告了肠杆菌属 Px6-4 FADase 及其与底物类似物复合物的晶体结构。我们的分析表明，FADase 具有半开口的底部β-桶，其催化口袋位于核心β-桶的中部和螺旋底部之间。其结构与酚酸脱羧酶（PAD）超家族的成员具有高度相似性。结构分析表明，FADase 通过涉及酶表面 8×8×15Å 尺寸口袋的“开-关”机制催化反应。活性口袋可以直接与溶剂接触，并在底物类似物诱导时允许底物进入。定点突变显示，E134A 突变使酶活性降低超过 60%，而 Y21A 和 Y27A 突变完全使酶失活。综合结构和突变结果表明，在 FADase 脱羧阿魏酸过程中，Trp25 和 Tyr27 对于底物的进入和正确取向是必需的，而 Glu134 和 Asn23 参与质子转移。
