Purification and characterization of NAD-dependent morphine 6-dehydrogenase from hamster liver cytosol, a new member of the aldo-keto reductase superfamily

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine t o morphine, was purified 815-fold to a homogeneous protein from the soluble fraction of hamster liver with a yield of 15%. The enzyme was a monomeric protein with a molecular weight of 38 kDa and an isoelectric point of 5.6. Although both NAD and NADP served as cofactors, the enzyme activity with NA DP was less than 5% that found with NAD at pH 7.4, With NAD, the enzyme gav e the maximal activity at pH 9,3, and the K-m and V-max values toward morph ine were 1.0 mM and 0.43 unit/mg protein, respectively. Among morphine cong eners, normorphine exhibited higher activity than morphine, but codeine and ethylmorphine were poor substrates, and dihydromorphine and dihydrocodeine showed no detectable activity. The enzyme also exhibited significant activ ity for a variety of cyclic and alicyclic alcohols. In addition to xenobiot ics, the enzyme catalyzed the dehydrogenation of 17 beta-hydroxy-steroids w ith much higher affinities than morphine. In the reverse reaction, the enzy me exhibited high activity for o-quinones, but morphinone, naloxone, and ar omatic ic aldehydes and ketones were reduced at slow rates, Sulfhydryl reag ents and ketamine strongly inhibited the enzyme, whereas pyrazole, barbital , and indomethacin had little effect on enzyme activity. 17 beta-Hydroxyste roids inhibited the enzyme in a competitive manner against morphine. A tota l of 302 amino acid residues, which comprised approximately 94% of whole pr otein, were identified by sequencing of the peptides obtained by proteolyti c digestion. This amino acid sequence of the enzyme showed significant homo logy to members of the aldo-keto reductase (AKR) superfamily and shared 63- 64% identity with members of the AKR1C subfamily. These findings indicate t hat the enzyme is a new member of the AKR superfamily that is involved in s teroid metabolism as 17 beta-hydroxysteroid dehydrogenase as well as xenobi otic metabolism. (C) 2000 Academic Press.