Functionalization of aromatic compounds by decarboxylases catalyzing regioselective carboxylation

催化氧化或非氧化脱酸反应的各种酶参与微生物中芳香族羧酸的好氧或厌氧降解。最近，我们发现并鉴定了两种新型的脱羧酶，它们能有效地催化逆羧化反应。这些发现有力地表明，普遍存在一个可逆催化芳香族羧酸盐脱羧基的酶群。在本研究中，我们考察和鉴定了其他可逆脱羧酶，并将其应用于芳香族羟基羧酸的合成。我们发现了一株细菌，Pandoraea sp.12B-2，其全细胞不仅催化2，6-二羟基苯甲酸酯的脱羧基，而且还催化1，3-二羟基苯的区域选择性羧化为2，6-二羟基苯甲酸酯。细菌全细胞还催化苯酚和1，2-二苯酚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。羧化反应的摩尔转化率依赖于反应混合物中KHCO3的浓度。在3MKHCO3存在下，约50%的1，3-二羟基苯被转化为2，6-二羟基苯甲酸酯。2，6-二羟基苯甲酸酯的产率达到了1.43M，是迄今为止报道的最高值。分离和克隆了Pandoraea sp.12B-2和根癌农杆菌IAM12048的2，6-二羟基苯甲酸脱羧酶基因。推测的脱羧酶一级结构与已报道的各种脱羧酶和羧基酶没有同源性。对一级结构的进一步分析表明，可逆脱羧酶分为两个亚群，结构，各种可逆脱羧酶分为两个亚群。

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.