Carbonyl reducing
enzymes play important roles in the biotransformation and detoxification of endo- and
xenobiotics. They are grouped into two
protein superfamilies, the
short-chain dehydrogenases (SDR) and
aldo-keto reductases (AKR), and usually are present in the cytoplasm of a cell. So far, only one membraneous
carbonyl reductase has been described,
11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is located in the endoplasmic reticulum and which significantly contributes to the metabolism of a variety of carbonyl containing drugs and toxicants.
Oracin is a new and prospective anticancer
drug bearing a prochiral carbonyl moiety. The main metabolic pathway of
oracin is carbonyl reduction to
11-dihydrooracin (DHO) which, however, eliminates the therapeutic potential of the
drug, because the two DHO enantiomers formed have significantly less anti-
tumor activities. Therefore, the
oracin inactivating
enzymes should urgently be identified to search for specific inhibitors and to enhance the chemotherapeutic efficacy. Interestingly, the calculation of
enzyme specific activities and stereospecificities of (+)-DHO and (-)-DHO formation strongly suggested the existence of a second, hitherto unknown microsomal
oracin carbonyl reductase in human liver. Therefore, the aim of the present study was to provide proof for the existence of this new
enzyme and to develop a purification method for further characterization. First, we succeeded in establishing a gentle solubilization technique which provided a favourable
detergent surrounding during the further purification procedure by stabilizing the native form of this fragile
protein. Second, we could partially purify this new microsomal
carbonyl reductase by a two step separation on Q-
sepharose followed by
Phenyl-sepharose. The
enzyme turned out to be
NADPH specific, displaying kinetic values for
oracin carbonyl reduction of K(m)=42 microM and V(max)=813 nmol/(30 min x mg
protein). Compared to the microsomal fraction, the
enzyme specific activity towards
oracin could be enhanced 73-fold, while the stereospecificity of (+)-DHO formation shifted from 40% to 86%. Considering these data for
11beta-HSD1, as described in previous reports, it is clear that the microsomal
carbonyl reductase investigated in the present study is new and has a great potential to significantly impair the
chemotherapy with the new anticancer
drug oracin.