Functionalization of aromatic compounds by decarboxylases catalyzing regioselective carboxylation

各种催化氧化或非氧化脱羧反应的酶参与了微生物对芳香羧酸的好氧或厌氧降解。最近，我们发现并表征了两种新型脱羧酶，它们有效地催化了反羧化反应。研究结果强烈表明，普遍存在一种可逆催化芳香羧酸脱羧的酶群。在本研究中，我们调查并表征了其他可逆脱羧酶，并将其应用于芳香族羟基羧酸的合成。我们发现了一种细菌Pandoraea sp.12B-2，它的整个细胞不仅催化2,6-二羟基苯甲酸酯的脱羧，而且还催化1,3-二苯酯的区域选择性羧化为2,6-二羟基苯甲酸酯。细菌的整个细胞还催化了苯酚和1,2-二羟基苯的区域选择性羧化反应，分别生成4-羟基苯甲酸酯和2,3-二羟基苯甲酸酯。羧基化反应的摩尔转化率取决于反应混合物中KHCO_3的浓度。加入的1,3-二羟基苯约50%在3 M KHCO_3存在下转化为2,6-二羟基苯甲酸酯。优化2,6-二羟基苯甲酸酯的高效产率后，2,6-二羟基苯甲酸酯的产率达到1.43 M，是迄今报道的最高值。羧基化反应后未观察到任何其他产物的形成。分离并克隆了Pandoraea sp.12B-2和农杆菌IAM12048的2,6-二羟基苯甲酸脱羧酶基因。推导出的脱羧酶一级结构与文献报道的各种脱羧酶和羧化酶没有同源性。进一步的一级结构分析表明，可逆脱羧酶可分为两个亚群，结构上，各种可逆脱羧酶可分为两个亚群。

Various enzymes catalyzing oxidative or nonoxidative decarboxylation reaction are involved in aerobic or anaerobic degradation of aromatic carboxylic acids in microorganisms. Recently, we found and characterized two kinds of novel decarboxylase, catalyzing the reverse carboxylation reaction efficiently. The findings strongly suggested the universal occurrence of an enzyme group reversibly catalyzing the decarboxylation of aromatic carboxylates. In the present study, we surveyed and characterized other reversible decarboxylases, and then applied them to the synthesis for aromatic hydroxycarboxylic acids.We found a bacgerium, Pandoraea sp.12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of the bacterium also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO_3 in the reaction mixture. About 50% of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 3 M KHCO_3. When the efficient production of 2,6-dihydroxybenzoate was optimized, the. productivity of 2,6-dihydroxybenzoate reached to 1.43 M, which was he highest value so far reported. No formation of any other products was observed after the carboxylation reaction.The genes encoding 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp.12B-2 and Agrobacterium tumefaciens IAM12048 were isolated and cloned. The deduced primary structures of the decarboxylases showed no homology with various decarboxylases and carboxylases reported. Further analysis on primary structures demonstated that reversible decarboxylases were classified into two subgroups, structures, various reversible decarboxylase were classified into two subgroups.