A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp strain QT12
A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp strain QT12
复制标题
编码 3-氨基苯甲酸 6-单加氧酶的新基因，参与丛毛单胞菌 QT12 菌株中 3-氨基苯甲酸的降解
DOI：
10.1007/s00253-018-9015-4
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发表时间：
2018-06-01
影响因子：
5
通讯作者：
Guo, Lizhong
中科院分区：
文献类型：
--
作者：
Yu, Hao;Zhao, Shuxue;Guo, Lizhong
3-氨基苯甲酸酯的生物降解途径已有文献记载，但对所涉及的蛋白质的序列和生化性质知之甚少。在本研究中，在降解3-氨基苯甲酸的Comamonas sp.中发现了一个与3-氨基苯甲酸降解有关的10,083个碱基的DNA片段。菌株QT12。在该DNA片段上发现了MABA基因，其编码的蛋白与NANA2的3-羟基苯甲酸-6-羟基酶有39%的氨基酸序列同源性，该突变株不能生长和转化3-氨基苯甲酸。在大肠杆菌中异源表达了MABA，纯化后得到了与48 kDa His标记蛋白相似的同源蛋白。该酶的性质为3-氨基苯甲酸-6-羟基酶，能催化3-氨基苯甲酸转化为5-氨基水杨酸，并在产物中引入一个氧原子。它含有一个非共价但紧密结合的FAD作为假体基团，NADH作为外部电子供体。在等摩尔NADH用量下生产5-氨基水杨酸酯。纯化后的酶对3-氨基苯甲酸的表观K-m和k(CAT)分别为158.51+/-4.74mU M和6.49+/-0.17 S(-1)，对NADH的表观K-m和k(CAT)值分别为189.85+/-55.70mU M和7.41+/-1.39 S(-1)。结果表明，MABA是菌株QT12降解3-氨基苯甲酸的必需底物，而3-氨基苯甲酸是MABA的主要生理底物。
The biodegradation pathway of 3-aminobenzoate has been documented, but little is known about the sequence and biochemical properties of the proteins involved. In the present study, a 10,083-bp DNA fragment involved in 3-aminobenzoate degradation was identified in 3-aminobenzoate-degrading Comamonas sp. strain QT12. The mabA gene, whose encoded protein shares 39% amino acid sequence identity with 3-hydroxybenzoate 6-hydroxylase of Polaromonas naphthalenivorans CJ2, was identified on this DNA fragment, and the mabA-disrupted mutant was unable to grow on and convert 3-aminobenzoate. MabA was heterologously expressed in Escherichia coli and purified to homogeneity as an approximately similar to 48-kDa His-tagged protein. It was characterized as 3-aminobenzoate 6-hydroxylase capable of catalyzing the conversion of 3-aminobenzoate to 5-aminosalicylate, incorporating one oxygen atom from dioxygen into the product. It contains a non-covalent but tightly bound FAD as the prosthetic group and NADH as an external electron donor. 5-Aminosalicylate was produced with equimolar consumption of NADH. The apparent K-m and k(cat) values of the purified enzyme for 3-aminobenzoate were 158.51 +/- 4.74 mu M and 6.49 +/- 0.17 s(-1), respectively, and those for NADH were 189.85 +/- 55.70 mu M and 7.41 +/- 1.39 s(-1), respectively. The results suggest that mabA is essential for 3-aminobenzoate degradation in strain QT12, and that 3-aminobenzoate is the primary and physiological substrate of MabA.