Abstract
Background17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions.ResultsWe have investigated here which secondary structure elements are involved in the dimerization of 17β-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 αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-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.ConclusionWe have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF 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.
Highlights
Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit
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
Summary
Preparation of mutant proteins The mutant proteins were prepared using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) and the pGex-17β-HSDcl expression vector [10]. The following primers were used (only mutagenic forward primers are shown; the mutations introduced are underlined): Arg129Asp for: 5'-GCCTCAACACCGACGGCCAGTTCTTCG-3' His111Ala recorded at 20°C at 0.1 nm increments between 250 and 197 nm using 0.1 cm path length cuvettes. The scanning speed was 50 nm/min, the response time 1 sec, and an average of 4 scans was recorded. The protein concentrations were approximately 0.2 mg/ml, in PBS (pH 7.3). Expression and purification of proteins All of the proteins were expressed as GST-fusion proteins in the JM107 E. coli strain and purified by affinity chromatography on Glutathione Sepharose followed by cleavage with thrombin, as described previously [10]. SDS PAGE Homogeneity of the proteins was checked by SDS-PAGE. Samples (4 μg) were denatured in Laemmli sample buffer (5 min at 90°C), applied to 12% acrylamide gels, and visualized by Coomassie Blue staining
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