Reductase Activity On Vitamin K3 Of Rabbit NADP(H)-Dependent Retinal Dehydrogenase/Reductase Expressed In Escherichia Coli

Retinoic acid (RA), a physiologically active compound in many vertebrates, has extensive biological functions in vivo, including cell growth, cell differentiation, morphogenesis and immunologic regulatory function. RA can be derived in vivo from retinol in two oxidative steps with retinal as an intermediate.The change from retinol to retinal is the rate-limited step in these reactions.NADP(H)-Dependent Retinol Dehydrogenase/Reductase (NRDR) identified from rabbit liver cytosol is involved in oxidoreduction between retinol and retinal with high activity. Therefore, NRDR plays an important role in modulation of the retinoids and RA homeostasis. NRDR can not only reduce retinal to retinol but also show relatively broad reductive activities to some other aldoketones. We believe that NRDR might play some other important functions. Vitamin K3(menadione), which contains two ketone groups at the 1,4-position of the naphthoquinone nucleus, might be a potential substrate of NRDR. Vitamin K3 is the endogenous precursor of the Vitamin K1 (phylloquinone) and Vitamin K2(menaquinones), and plays an important role in blood coagulation. In this study, we aimed to investigate the function of NRDR and its possible reductase activity of om Vitamin K3.An expression vector containing the full sequence of the Rabbit-NRDR was constructed and transformed into E. coli BL21-AI. The supernatant fractions were obtained after sonication and centrifugation. 6 His-tagged recombinant Rabbit-NRDR protein was harvested by affinity chromatography, and its identity was confirmed by SDS-PAGE analysis and activity determination. Oxydoreductase activity of the recombinant Rabbit-NRDR for other potential substrates such as 9,10-Phenanthrenequinone, phosphopyridoxal and prostaglandin E2, were detected. It is found by chemical synthesis, high performance liquid chromatography (HPLC) and fluorescence spectra that Rabbit-NRDR could catalyze Vitamin K3 to Vitamin K4 (menadiol). Under hypoxic conditions and the presence of SDS, enzyme kinetics study was carried out using high performance liquid chromatography (HPLC), and the values of the Km and Vmax obtained were 30.70μmol and 9.091μmol/(min·mg) respectively. Having not very low values of Km suggested that NRDR may also play some physiological roles in the III metabolism of vitamin K in vivo.