Background: 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17beta-HSDcl is also a homodimer under native conditions.
Results: We have investigated here which secondary structure elements are involved in the dimerization of 17beta-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the alphaE-helices interact with the Asp121, Glu117 and Asp187 residues from the alphaE and alphaF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17beta-HSDcl monomeric, while the mutant 17beta-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity.
Conclusion: We have shown by site-directed mutagenesis and structure/function analysis that 17beta-HSDcl dimerization involves the alphaE and alphaF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.