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

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K_m值为0.082mmol/L，V_max值为56.26±0.02 μmol/（min·mg）；5min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。

Abstract:

[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 araC 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 and β-hydroxysteroid dehydrogenase. 17β-HSD2 oxidized E2 at C₁₇ site using NAD⁺ as cofactor, with 0.0802 mmol/L K_m value and 56.26±0.02 μmol/(min·mg) V_max 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.