Molecular and Biochemical Characterizations of Dehydroascorbate Reductase from Sesame (Sesamum indicumL.) Hairy Root Cultures

Abstract

Dehydroascorbate reductase (DHAR) is a biotechnologically or physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction for most higher plants. A DHAR cDNA was isolated from sesame (Sesamum indicum L.) hairy roots, and its structure and biochemical properties were characterized to provide some information about its expressional and biochemical profiles in the hairy root cultures. The cDNA contained a catalytic motif CXXS, which may be indicative of a thiol-dependent redox function. A fusion DHAR expressed in an Escherichia coli expression system was purified with four purification steps until a homogeneous single band signal was seen in an acrylamide gel, and its antibody was prepared for Western blot analyses. The biochemical results showed that the purified recombinant DHAR had an optimal pH of around 6.0, which was different from those (pH 7.8-8.2) of other plant species. The temperature optimal for the DHAR activity was in a relatively wide range of 30-60 degrees C. It was proved by a real-time RT-PCR technique that the transcription activity of the DHAR was about 2-5-fold higher during the first 3 week cultures than during the latter 3 week ones. The highest activity of the sesame DHAR was detected in the 4 week cultures of the hairy roots, after which its activity was rapidly decreased to approximately 80%, suggesting that the most active DHAR occurred in this culture period. Western blot analyses confirmed that the presence of DHAR enzyme was identified in both cultures of the fused E. coli and the sesame hairy roots.

Effect of Cooking and Reconstitution Methods on the Loss of Bioactive Compounds in Pigmented and Unpigmented Potatoes

“…The coloured potatoes had 1.7 to 2.5 times more the phenolics presumably due to the high anthocyanins in them and about 2 to 3 times more antioxidant capacity compared to unpigmented genotypes. These findings are in consistence with [5] [17] [26] and [27] who also reported greater amounts of bioactive compounds in coloured potatoes. No anthocyanins were detected in the unpigmented potatoes however; coloured genotypes had higher levels of anthocyanins.…”

“…One of the environmental signals that activate tuberization in potatoes is light. Delivery of light from the light-receiving organ, the leaf, to an underground organ growing in the dark, the stolon, leads to the formation of potato tuber [27] [36]. However, the underground tubers are exposed to darkness while the photosynthetically active leaf tissues are exposed to the light.…”

Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

“…Native PAGE showed that the molecular weight of the recombinant PbDHAR was less than 43 kDa (Figure 4C). These results strongly indicate that the recombinant PbDHAR is a monomer protein of 29.69 kDa, which is similar to other plant DHARs (Shimaoka et al 2000; Dixon et al 2002; Chun et al 2007).…”

Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plant and human

“…Further, structure-guided sequence alignment reveals a distinct positively charged pocket shared by these proteins [33] (Figure 1c, d). This pocket is occupied by conserved amino acids distributed around the catalytic CXX[C/S] motif [34]. In addition, the following residues; K8, F22, V60, P61, D73 and S74 which are part of the positively charged pockets of PgDHAR are also conserved in HsCLIC1 as well (K13, F26, L64, P65, D76 and T77) (Figure 1d).…”