Characterization of a LuxR repressor for 3,17ß-HSD in Comamonas testosteroni ATCC11996.

ABSTRACT

3,17ß-Hydroxysteroid dehydrogenase in Comamonas testosteroni (C. testosteroni) is a key enzyme involved in the degradation of steroid compounds. Recently, we found that LuxR is a negative regulator in the expression of the 3,17ß-HSD gene. In the present work, we cultured wild-type and LuxR knock-out mutants of C. testosteroni with inducers such as testosterone, estradiol, progesterone or estrone. HPLC analysis showed that the degradation activities towards testosterone, estradiol, progesterone, and estrone by C.T.-LuxR-KO1 were increased by 7.1%, 9.7%, 11.9% and 3.1%, respectively compared to the wild-type strain. Protein conformation of LuxR was predicted by Phyre 2 Server software, where the N-terminal 86(Ile), 116(Ile), 118(Met) and 149(Phe) residues form a testosterone binding hydrophobic pore, while the C-terminus forms the DNA binding site (HTH). Further, luxr point mutant plasmids were prepared by PCR and co-transformed with pUC3.2-4 into E. coli HB101. ELISA was used to determine 3,17ß-HSD expression after testosterone induction. Compared to wild-type luxr, 3,17ß-HSD expression in mutants of I86T, I116T, M118T and F149S were decreased. The result indicates that testosterone lost its capability to bind to LuxR after the four amino acid residues had been exchanged. No significant changes of 3,17ß-HSD expression were found in K354I and Y356 N mutants compared to wild-type luxr, which indicates that these two amino acid residues in LuxR might relate to DNA binding. Native LuxR protein was prepared from inclusion bodies using sodium lauroylsarcosinate. Molecular interaction experiments showed that LuxR protein binds to a nucleotide sequence which locates 87 bp upstream of the ßhsd promoter. Our results revealed that steroid induction of 3,17ß-HSD in C. testosteroni in fact appears to be a de-repression, where testosterone prevents the LuxR regulator protein binding to the 3,17ß-HSD promoter domain.