Cloning and molecular characterization of selenocysteine methyltransferase (AchSMT) cDNA from Astragalus chrysochlorus

Abstract

Selenium (Se) is a dietary essential trace element in human nutrition and has been implicated in health, including cancer preventation. Methylselenocysteine (MeSeCys) is an effective chemopreventative compound. Astragalus sp. are known to accumulate Se with the majority of the selenoamino acids in the form of MeSeCys. The aim of this study was the cloning and charactherization of a cDNA encoding selenocysteine methyltransferase (SMT), the key enzyme responsible for MeSeCys formation, from Astragalus chrysochlorus using specific primers. Our results showed that Astragalus chrysochlorus SMT (AchSMT) is a 1020 bp (base pair) cDNA (GQ844862) with an open reading frame predicted to encode a 339 amino acid, 36.94 kDa protein. The predicted amino acid sequence of AchSMT (AEI53593) revealed 97% identity with A. bisulcatus selenocysteine methyltransferase (AbSMT). Bioinformatic analysis revealed that AchSMT lacks chloroplast and mitochondrial targeting sequences. The AchSMT possess a possible zinc-binding motif (GGCC) and a conserved cysteine residue upstream of this motif AchSMT was expressed in Escherichia coli, then confirmed by DNA sequence analysis, western blotting and enzyme activity. Expression of AchSMT correlated with the presence of SMT enzyme activity in cell extracts. In A. chrysochlrous plants, analysis of AchSMT gene expression in response to selenate treatments showed that the AchSMT was constitutively expressed. The isolation of SMT gene will result in further studies for overproduction of valuable metabolite, methylselenocysteine.