Characterization and recombinant expression of the translational repressor RepB of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase in Comamonas testosteroni.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The gene of 3alpha-HSD/CR (hsdA) was cloned and characterized by our group. We have also reported that two repressor proteins (RepA and RepB) have been identified which regulate hsdA expression. To further characterize RepB, the protein was expressed in Escherichia coli and purified in an active state. Gel shift experiments showed that RepB binds to a 16 nucleotide sequence downstream of AUG of the hsdA mRNA, providing evidence that RepB acts on the translational level. The addition of testosterone to the culture medium led to a derepression. Furthermore, a plasmid was prepared containing a point mutation that inactivates only repA, but has no effect on hsdA, with which it happens to partly overlap. The result of coexpression experiments with this construct and a plasmid containing the genetic information for RepB showed that RepB is still active and is therefore not dependent on a functional RepA. In conclusion, RepB is a novel regulatory protein that inhibits the translation of hsdA mRNA, thereby leading to a decreased expression of 3alpha-HSD/CR.