Site-directed mutagenesis studies of bovine liver cytosolic dihydrodiol dehydrogenase: the role of Asp-50, Tyr-55, Lys-84, His-117, Cys-145 and Cys-193 in enzymatic activity

A previous report on the cloning, bacterial expression and purification of bovine liver cytosolic dihydrodiol dehydrogenase (DD3) cDNA (1,330 bp in fu ll length) using pKK223-3 expression vector characterize the properties of the recombinant DD3 in the aspects of substrate specificity and inhibitor s ensitivity (Terada et al., Adv. Exp. Biol. Res. 414 (1997) 543-53). The nuc leotide sequence of this DD3 cDNA completely matches that of bovine liver-t ype prostaglandin F synthase (PGFS) (Suzuki et al., J. Biol. Chem. 274 (199 9) 241-8). In the present study, a large amount of recombinant DD3 (rDD3) w as expressed in Escherichia coli BL21 (DE3) with a pET28a expression vector . The recombinant DD3 (rDD3) was easily and quickly purified to an apparent homogeneity with one step column chromatography of Ni2+-affinity resin. Th e rDD3 showed essentially the same substrate specificity and inhibitor sens itivity as purified liver DD3 (DD3). To analyze the role of amino acid resi dues of DD3 in its enzymatic activity, site-directed mutagenesis of DD3 wit h PCR method was performed. The results of the analyses of these mutants in the aspects of substrate specificity and cofactor-binding suggested a vari ety of functions in the enzymatic activity: as an active site Tyr-55 may ac t as a general acid and Asp-50, Lye-84 and His-117 may play an important ro le in the control of protonation of Tyr-55 as a general acid in the dehydro genase activity under higher pH conditions, though these residues may not b e involved in reductase activity under lower pH conditions. Though the muta ted DD3s (Cys to Ser! did not show significant differences in their substra te specificities, these mutants showed different sensitivities to SH-reagen ts. Present results indicate that Cys-193 may play an important role in the modulation of enzymatic activity under redox conditions generated with GSH + GSSG among five cysteines in DD3. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.