Characterization Of Hydroxysteroid Dehydrogenase Responsible For 17?-estradiol Transformation And Identification Of Its Transcriptional Regulators In Pseudomonas Putida SJTE-1

Hydroxysteroid dehydrogenase?HSD?,the oxidoreductase dependent on NAD?P?+/NAD?P?H,belongs to the family of Short chain dehydrogenase/reductase?SDR?.HSDs are prevalent in plants and animals,and have been proved to be a key enzyme for hormone metabolism in animals.Pseudomonas putida SJTE-1 has been comfirmed to be capable of degrading estrogens like 17?-estradiol efficiently.Genome sequence and comparative genomic analysis showed there existed several potential enzymes involved in the transformation of estrogens in this strain.However,the enzymatic properties and its regulatory factors are still unclear.This work focused on the two short chain dehydrogenase/reductase?HSD₁172 and ACP reductase₄227?and characterized their enzymatic properties;two regulators involved in their transcription were also identified.The results of this work could promote the mechanism study on bacterial degradation of estrogens.Results showed that two short chain dehydrogenase/reductase?HSD₁172and ACP reductase₄227?were effectively induced when strain SJTE-1 was cultured with estrogen-derived carbon.Protein sequence alignment and phylogenetic tree results showed that both of the two enzymes were17?-steroid dehydrogenase?17?-HSD?,which can catalyze the reduction of ketone or the oxidization of hydroxyl at the C17 site of the substrate.Further the recombinant HSD₁172 and ACP reductase₄227 proteins were obtained by heterologous expression and affinity purification,with protein sizes of25.56 Kd and 26.91 Kd.The two proteins both used NAD⁺as their cofactor for the dehydrogenation of C17 site in estrogen D ring,and transform17?-estradiol?E2?into estrone?E1?.Their optimal reaction temperature was37?and the optimum pH was 9.0.Ca²⁺,Zn²⁺and Cu²⁺could inhibit the oxidation reaction,while Mg²⁺,Mn²⁺and Ni²⁺could promote the reaction.The K_m values of 17?-HSD₁172 and ACP reductase₄227 were 0.068 mM and 0.071 mM,respectively,and the V_maxax was 0.871 mM/s and 0.347 mM/s,respectively.Therefore,17?-HSD₁172 showed higher conversion efficiency for estradiol and played an important role in its degradation.Furthermore,two transcription factor proteins OxyR_like and CrgA_like probably involved in the regulation of 17?-HSD₁172 were identified.Sequence alignment and secondary structure analysis showed that they contained similar DNA binding structure with the OxyR and CrgA family regulators,which could bind the upstream promoter region of target gene and regulate gene transcription by identifying the conservative sequences T{N₁₁}A and GATA.After heterologous expression and affinity purification,two recombinant proteins were obtained.In vitro electrophoretic mobility shift assay?EMSA?showed that both OxyR_like protein and CrgA_like protein could bind to the upstream promoter region of hsd₁172 efficiently;and the binding of OxyR_like to DNA could be effectively dissociated by estradiol,while the binding of CrgA_like could not.Transcription and expression analysis indicated that both of the two genes could be induced by estradiol and involved in the regulation 17?-HSD₁172.In conclusion,the results of this project will promote the research on the molecular mechanism and regulation mechanism of the bacterial degradation of estrogen,and provide theoretical basis for the construction of efficient environmental estrogen-degrading engineering strains.