Dehydroascorbate reductase and monodehydroascorbate reductase activities of two metallothionein-like proteins from sweet potato (Ipomoea batatas [L.] Lam. 'Tainong 57') storage roots.

BACKGROUND

Metallothionein (MT) is a group of proteins with low molecular masses and high cysteine contents, and it is classified into different types, which generally contains two domains with typical amino acid sequences.

RESULTS

In this report, two full-length cDNAs (MT-1 and MT-II) encoding MT-like proteins were isolated from the roots of sweet potato (Ipomoea batatas [L.] Lam. 'Tainong 57'). Their open reading frames contained 642 and 519 nucleotides (66 and 81 amino acids) for MT-1 and MT-II, respectively, and exhibited a relatively low amino acid sequence similarity. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that MT-I is a member of the plant MT Type-I family, and MT-II is a member of the plant MT Type-II family. The corresponding mRNA levels of MT-1 and MT-II were the highest found in the storage roots. Recombinant MT-1 and MT-II protein overproduced in E. coli (M15) was purified by Ni2+-chelated affinity chromatography. MT-1 and MT-II reduced dehydroascorbate (DHA) in the presence of glutathione (GSH) to regenerate L-ascorbic acid (AsA). However, without GSH, MT-1 and MT-II has very low DHA reductase activity. And AsA was oxidized by AsA oxidase to generate monodehydroascorbate (MDA) free radical. MDA was also reduced by MT-1 and MT-II to AsA in the presence of NADH mimicking the MDA reductase catalyzed reaction.

CONCLUSIONS

These data suggest that MT-1 and MT-II have both DHA reductase and MDA reductase activities. MT-1 and MT-II are apparently the first reported plant MTs exhibiting both DHA and MDA activities in vitro.