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

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.