BINDING AND REACTIVITY OF CANDIDA-ALBICANS ESTROGEN-BINDING PROTEIN WITH STEROID AND OTHER SUBSTRATES

In this report recombinant estrogen binding protein (EBP1), isolated o riginally from Candida albicans as a result of its high affinity for 1 7 beta-estradiol, has been purified extensively using a modified affin ity purification scheme originally developed for a homolog of EBP1, ol d yellow enzyme (OYE). It is shown that like OYE, the protein binds a variety of compounds with a phenolic structure, including 17 beta-estr adiol, and compounds with an alpha,beta-unsaturated keto or aldehyde s tructure. In addition, EBP1 exhibits an NADPH oxidoreductase activity, transferring electrons from NADPH to all alpha,beta-unsaturated keton es and aldehydes tested via the tightly bound FMN cofactor. Analysis o f the steady-state kinetics of these reactions indicate a tetra uni pi ng-pong mechanism. Inhibition of the steady-state reaction by 17 beta- estradiol gives a K-i = 10 +/- 2 nM, and indicates exclusive binding o f this steroid to the enzyme in its oxidized state. In contrast, 19-no rtestosterone binds to both oxidized and reduced forms of the enzyme w ith dissociation constants of 600 +/- 100 and 650 +/- 90 nM, respectiv ely. EBP1 also catalyzes a disproportionation reaction with certain co mpounds, in which two molecules of a cylic alpha,beta-unsaturated keto ne, including the steroid 19-nortestosterone, are individually aromati zed and reduced to the corresponding saturated ketone. Despite the ext ensive similarity in sequence and enzymic activity, notable difference s between EBP1 and the OYE family of proteins exist with regard to the binding behavior and reactivity with the two steroids tested here, es tradiol and 19-nortestosterone.