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Characterization of 17β-hydroxysteroid dehydrogenase and regulators involved in estrogen degradation in Pseudomonas putida SJTE-1

Wang, Pingping, Zheng, Daning, Peng, Wanli, Wang, Yanqiu, Wang, Xiuli, Xiong, Weiliang, Liang, Rubing
Applied microbiology and biotechnology 2019 v.103 no.5 pp. 2413-2425
Pseudomonas putida, bacteria, biodegradation, enzyme activity, enzymes, estradiol, estrone, gene overexpression, genes, metabolism, promoter regions
In bacteria, the enzyme catalyzing the transformation of 17β-estradiol is considered the key enzyme for its metabolism, whose enzymatic activity and regulatory network influence the biodegradation efficiency of this typical estrogen. In this work, a novel 17β-hydroxysteroid dehydrogenase (17β-HSD) was characterized from the estrogen-degrading strain Pseudomonas putida SJTE-1, and two regulators were identified. This 17β-HSD, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, could be induced by 17β-estradiol and catalyzed the oxidization reaction at the C₁₇ site of 17β-estradiol efficiently. Its Kₘ value was 0.068 mM, and its Vₘₐₓ value was 56.26 μmol/min/mg; over 98% of 17β-estradiol was oxidized into estrone in 5 min, indicating higher efficiency than other reported bacterial 17β-HSDs. Furthermore, two genes (crgA and oxyR) adjacent to 17β-hsd were studied which encoded the potential CrgA and OxyR regulators. Overexpression of crgA could enhance the transcription of 17β-hsd, while that of oxyR resulted in the opposite effect. They could bind to the specific and different sites in the promoter region of 17β-hsd gene directly, and binding of OxyR could be released by 17β-estradiol. OxyR repressed the expression of 17β-hsd by its specific binding to the conserved motif of GATA-N₉-TATC, while CrgA activated the expression of this gene through its binding to the motif of T-N₁₁-A. Therefore, this 17β-HSD transformed 17β-estradiol efficiently and the two regulators regulated its expression directly. This work could promote the study of the enzymatic mechanism and regulatory network of the estrogen biodegradation pathway in bacteria.