Purification and Initial Characterization of 3-Hydroxybenzoate 6-Hydroxylase From a Halophilic Martelella Strain AD-3
文献类型： 外文期刊
作者： Chen, Xin 1 ; Tang, Hongzhi 2 ; Liu, Yongdi 1 ; Xu, Ping 2 ; Xue, Yong 4 ; Lin, Kuangfei 1 ; Cui, Changzheng 1 ;
作者机构： 1.East China Univ Sci & Technol, Sch Resources & Environm Engn, State Environm Protect Key Lab Environm Risk Asse, Shanghai, Peoples R China
2.Shanghai Jiao Tong Univ, State Key Lab Microbial Metab, Shanghai, Peoples R China
3.Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai, Peoples R China
4.Shanghai Acad Agr Sci, Ecoenvironm Protect Res Inst, Shanghai, Peoples R China
关键词： moderate halophilic bacteria; 3-hydroxybenzoate; 3-hydroxybenzoate 6-hydroxylase; purification; FAD binding
期刊名称：FRONTIERS IN MICROBIOLOGY （ 影响因子：5.64； 五年影响因子：6.32 ）
ISSN： 1664-302X
年卷期： 2018 年 9 卷
页码：
收录情况： SCI
摘要： 3-Hydroxybenzoate 6-hydroxylase, an NADH-dependent flavoprotein, can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons. 3-Hydroxybenzoate is metabolized by entering the TCA cycle through the gentisate pathway. We found a putative 3HB6H gene from a cluster that potentially encodes for gentisic acid degradation from a halophilic Martelella sp. strain AD-3. The corresponding protein was expressed with an N-terminal His-tag and purified by Ni2+-nitrilotriacetic acid affinity chromatography. The protein showed an overexpressed band of about 46 kDa by SDS-PAGE, and it was also proven that the enzyme contains FAD by absorption spectroscopy and HPLC analysis. The optimal activity of 3HB6H from strain AD-3 was observed in phosphate buffer (pH 8.0) at 37 degrees C without salinity (NaCl) and metal salts. The K-m values of 3-hydroxybenzoate 6-hydroxylase were determined to be 72.6 +/- 10.1 mu M and 104.1 +/- 18.2 mu M for 400 mu M NADH and 3-hydroxybenzoate, respectively. Site-directed mutagenesis showed that residues 305, 306 and 308 are important for FAD binding. In addition, we found that Tyr221 and Gln305 of 3HB6H from strain AD-3 are involved in substrate binding.
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