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

Author:
Wang, Pingping, Zheng, Daning, Peng, Wanli, Wang, Yanqiu, Wang, Xiuli, Xiong, Weiliang, Liang, Rubing
Source:
Applied microbiology and biotechnology 2019 v.103 no.5 pp. 2413-2425
ISSN:
0175-7598
Subject:
Pseudomonas putida, bacteria, biodegradation, enzyme activity, enzymes, estradiol, estrone, gene overexpression, genes, metabolism, promoter regions
Abstract:
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.
Agid:
6327469