Mutational analyses of cysteine residues of bovine dihydrodiol dehydrogenase 3.
The cloning, bacterial expression and purification of bovine liver cytosolic dihydrodiol dehydrogenase 3 (DD3) cDNA (1330 bp in full length) using the pKK223-3 expression vector has been reported previously. Recombinant DD3 (rDD3) was characterized in terms of its substrate specificity and inhibitor sensitivity [Terada et al., Adv. Exp. Biol. Res. 414 (1997) 543-553]. The nucleotide sequence of DD3 cDNA completely matched with that of bovine liver-type prostaglandin F synthase [Suzuki et al., J. Biol. Chem. 274 (1999) 241-248]. In the present study, we succeeded in high level expression of rDD3 in Escherichia coli BL21 (DE3) using the pET28a expression vector. rDD3 was easily and quickly purified to apparent homogeneity by one-step column chromatography using Ni(2+)-affinity resin. Furthermore, rDD3 showed essentially the same substrate specificity and inhibitor sensitivity to that of purified liver DD3. To analyze the role of cysteines (145, 154, 188, 193 and 206) in the enzymatic activity of DD3, site-directed mutagenesis of DD3 using the polymerase chain reaction method was performed. Mutants (C145S, C154S, C188S, C193S and C206S) were analyzed for substrate specificity, cofactor binding and inactivation by disulfide (dithio-bis(2-nitrobenzoic acid), alkylating reagent (N-ethylmaleimide) and oxidants (naphthoquinone and H(2)O(2)) Results indicated that these five cysteines of rDD3 may not be directly involved in substrate or cofactor binding. Mutant C193S showed strong resistance to SH-reagents unlike wild-type DD3 (WT) or other mutants. Both the WT and the other mutants showed essentially the same sensitivity to SH-reagents. Cofactor (NADP(+)) protected mutants C145S, C188S and C206S from inactivation as well as WT, while NAD(+) was not protective. Our present results indicate that Cys193, which is located close to the NADP(+)-binding site, may be involved in the alteration of enzymatic activity.
Terada
T
T
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, 565-0871, Osaka, Japan. terada@phs.osaka-u.ac.jp
Fujita
N
N
Adachi
H
H
Nanjo
H
H
Sato
R
R
Takagi
T
T
Maeda
M
M
D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012694:Serine; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; D003545:Cysteine