Huang Yan, Zhao Ke-Xin, Shen Xi-Hui, Jiang Chen-Ying, Liu Shuang-Jiang
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Datun Road Jia-3#, Chaoyang District, Beijing, 100101, People's Republic of China.
Appl Microbiol Biotechnol. 2008 Feb;78(1):75-83. doi: 10.1007/s00253-007-1286-0. Epub 2007 Dec 11.
Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA ( Cg ) (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA (Cg) was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA(Cg). The pobA (Cg) is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.
谷氨酸棒杆菌利用4-羟基苯甲酸（4HBA）作为唯一碳源进行生长。先前的研究表明，4HBA通过PcaK被摄入细胞，芳香环通过原儿茶酸3,4-双加氧酶被裂解。在本研究中，鉴定了参与将4HBA转化为3,4-二羟基苯甲酸（原儿茶酸）的基因pobA（Cg）（ncgl1032），其基因产物PobA（Cg）被表征为一种4HBA 3-羟化酶，它是PobA（Cg）的同型二聚体。pobA（Cg）与pcaK在物理上相关联并形成一个假定的操纵子，但这两个基因与编码用于4HBA/原儿茶酸降解的其他酶的pca簇相距较远。这种新的4HBA 3-羟化酶的独特之处在于，尽管其序列与其他以NADH作为辅酶底物的4HBA 3-羟化酶相似，但它更倾向于以NADPH而非NADH作为辅酶底物。对假定的NADPH结合位点D38和T42进行定点诱变，进一步提高了其对NADPH的亲和力及其催化效率。
