Myrosinase in horseradish ( Armoracia rusticana) root: Isolation of a full-length cDNA and its heterologous expression in Spodoptera frugiperda insect cells

Abstract

Myrosinase (β-thioglucoside glucohydrolase, EC. 3.2.3.1), is the only known S-glucosidase in plants, catalyzing the hydrolysis of glucosinolates into compounds that have diverse biological activities. In the present study, a full-length cDNA encoding myrosinase ( ArMY1) was cloned from horseradish ( Armoracia rusticana) root. ArMY1 has an open reading frame of 1614 nucleotides with a deduced protein of 538 amino acids and molecular mass of 61.6 kD. ArMY1 shows highest overall amino acid identity (72%) with Arabidopsis thaliana myrosinase TGG2. Heterologous expression of ArMY1 in baculovirus-infected Sf9 cells resulted in an immunologically active recombinant ArMY1 protein, when probed with myrosinase-specific monoclonal antibody 3D7, with apparent mass 65 kD. ArMY1 mRNA signal of about 1.95 kb was detected in the leaves and roots of horseradish, but not in the florets of broccoli. Phylogenetic analysis showed that ArMY1 does not cluster with any of the currently known myrosinase subfamilies, MA, MB, MC, and may represent a novel myrosinase subfamily. This is the first report of cloning of myrosinase cDNA from horseradish root. It provides important sequence information that will enable further studies of myrosinase expression patterns and their interaction with myrosinase-binding proteins and other myrosinase-associated proteins.