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 full length) using pKK223-3 expression vector characterize the properties of the recombinant DD3 in the aspects of substrate specificity and inhibitor sensitivity (Terada et al., Adv. Exp. Biol. Res. 414 (1997) 543-53). The nucleotide sequence of this DD3 cDNA completely matches that of bovine liver-type prostaglandin F synthase (PGFS) (Suzuki et al., J. Biol. Chem. 274 (1999) 241-8). In the present study, a large amount of recombinant DD3 (rDD3) was 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 Ni(2+)-affinity resin. The rDD3 showed essentially the same substrate specificity and inhibitor sensitivity as purified liver DD3 (DD3). To analyze the role of amino acid residues of DD3 in its enzymatic activity, site-directed mutagenesis of DD3 with PCR method was performed. The results of the analyses of these mutants in the aspects of substrate specificity and cofactor-binding suggested a variety of functions in the enzymatic activity: as an active site Tyr-55 may act as a general acid and Asp-50, Lys-84 and His-117 may play an important role in the control of protonation of Tyr-55 as a general acid in the dehydrogenase activity under higher pH conditions, though these residues may not be involved in reductase activity under lower pH conditions. Though the mutated DD3s (Cys to Ser) did not show significant differences in their substrate specificities, these mutants showed different sensitivities to SH-reagents. 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.
Terada
T
T
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, 565-0871, Osaka, Japan. terada@phs.oskak-u.ac.jp
Fujita
N
N
Sugihara
Y
Y
Sato
R
R
Takagi
T
T
Maeda
M
M
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D017931:DNA Primers; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; D004229:Dithiothreitol