恶臭假单胞菌SJTE-1中氧化17β-雌二醇的17β-羟甾类脱氢酶2及其转录调控因子AraC的鉴定  

Characterization of 17β-hydroxysteroid dehydrogenase 2 and the AraC regulator involved in 17β-estradiol oxidization in Pseudomonas putida SJTE-1

作　　者：孙欣 郑达宁 彭万里 梁如冰[1] Xin Sun;Daning Zheng;Wanli Peng;Rubing Liang(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]微生物代谢国家重点实验室,上海交通大学生命科学技术学院,上海200240

出　　处：《微生物学报》2020年第2期306-319,共14页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31570099,31370152)。

摘　　要：[目的]假单胞菌SJTE-1可高效转化17β-雌二醇,但其代谢机制尚不清楚。本文鉴定和表征了该菌株中参与雌二醇降解与调控过程的17β-羟甾类脱氢酶2(17β-HSD2)和转录调控因子AraC。[方法]我们通过荧光定量PCR分析了17β-hsd2和araC的转录水平;我们在大肠杆菌BL21(DE3)菌株中异源表达了17β-HSD2和AraC基因,并利用金属离子亲和层析法纯化获得了重组蛋白;我们体外表征了17β-HSD2的酶学性质,利用高效液相色谱鉴定了其产物;通过电泳迁移转移法和DNase酶I足迹试验,我们鉴定了重组蛋白AraC的结合能力与结合位点。[结果]17β-HSD2和AraC可被17β-雌二醇诱导表达;蛋白序列比对结果表明17β-HSD2含有短链脱氢酶/还原酶(SDR)和β-羟甾类脱氢酶的保守结构与残基。该酶以NAD+为辅助因子,在C17位点氧化17β-雌二醇,其Km值为0.082 mmol/L,Vmax值为56.26±0.02μmol/(min·mg);5 min内可转化97.4%以上的雌二醇。转录调控因子AraC可直接结合17β-hsd2基因启动子区的特异位点;雌二醇与雌酮可解除这一结合,启动17β-hsd2基因转录;过表达AraC蛋白可抑制17β-hsd2的转录。[结论]假单胞菌SJTE-1的17β-羟甾类脱氢酶2可高效催化17β-雌二醇转化,并受到转录因子AraC的直接调控。本工作可推进细菌的雌激素降解酶学机制与调控网络研究。[Objective]Pseudomonas putida SJTE-1 can degrade17β-estradiol(E2)efficiently,but its degradation mechanism is still unclear.Here we characterized a 17β-hydroxysteroid dehydrogenase 2(17β-HSD2)and one AraC regulator responsible for oxidization and regulation of E2 biodegradation.[Methods]We detected the transcription of 17β-hsd2 and ara C by reverse transcription and quantitative PCR.We overexpressed 17β-HSD2 and AraC in Escherichia coli BL21(DE3)strain and purified them with metal-ion affinity chromatography.We characterized the enzymatic properties of 17β-HSD2 in vitro and detected the product with High Performance Liquid Chromatography.We determined the binding capability and binding sites of AraC by electrophoretic mobility shift assay and DNase I footprinting assay.[Results]Results showed the transcription of 17β-HSD2 and AraC were induced by E2.Multiple sequences alignment showed 17β-HSD2 contained the conserved structure and residues of short-chain dehydrogenase/reductase andβ-hydroxysteroid dehydrogenase.17β-HSD2 oxidized E2 at C17 site using NAD+as cofactor,with 0.0802 mmol/L Km value and 56.26±0.02μmol/(min·mg)Vmax value;over 97.4% of E2 was transformed into estrone in five minutes.AraC protein could directly bind to the specific sites in the promoter region of 17β-hsd2,which could be released by E2 or estrone.Overexpression of AraC repressed the transcription of 17β-hsd2 significantly.[Conclusion]17β-HSD2 catalyzed the transformation of 17β-estraiol efficiently and was regulated by AraC in P.putida SJTE-1.This work promoted the enzymatic mechanism and the regulatory network studies about bacterial estrogen biodegradation.

关 键 词：17β-羟甾类脱氢酶2 17Β-雌二醇 转录因子AraC 雌酮 生物降解 

分 类 号：Q933[生物学—微生物学]