PMID: 11343798May 10, 2001Paper

Mutational analyses of cysteine residues of bovine dihydrodiol dehydrogenase 3

Biochimica Et Biophysica Acta
T TeradaM Maeda

Abstract

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...Continue Reading

References

Jan 1, 1989·The International Journal of Biochemistry·H SawadaK Matsuura
Jan 1, 1993·Journal of Enzyme Inhibition·T TeradaT Nishihara
Oct 1, 1993·Chemico-biological Interactions·T M Penning
Oct 23, 1997·The Biochemical Journal·J M JezT M Penning
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Dec 29, 1998·The Journal of Biological Chemistry·T SuzukiK Watanabe
Aug 1, 1961·The Biochemical Journal·G N WILKINSON

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Citations

Jul 2, 2002·Nature Biotechnology·Gregory C AdamBenjamin F Cravatt

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