Characterization of an iron-induced enzyme, nicotianamine synthase, from giant leucaena

Abstract

Metal homeostasis in giant leucaena (Leucaena leucocephala subsp. glabrata) is of interest due to the plant’s production of mimosine, an iron-chelating secondary metabolite. Real-time PCR performed on root and foliar tissue showed the upregulation of 19 genes following exogenous application of iron. Notable genes affected include glutathione synthase (20-fold increase in leaf), ferric chelate reductase (15-fold increase in root), mimosinase (20-fold increase in leaf) and nicotianamine synthase (30-fold increase in root). Transcriptome sequence data and 5’-RLM-RACE methods identified the complete nicotianamine synthase coding sequence, which was cloned for heterologous expression and in vitro assays. To properly assay nicotianamine synthase activity, due to strong feedback inhibition by 5’-methylthoadenosine, the giant leucaena 5’-methylthoadenosine nucleosidase was cloned and purified as well. Additional inhibition produced by the substrate compound, S-adenosylmethionine (SAM), was discovered in this study by utilizing a recombinant SAM-synthetase. Nicotianamine synthase is sensitive to racemic mixtures of SAM, which is inevitably produced in commercial SAM solutions. When substrate was produced in situ, using SAM-synthetase, nicotianamine synthase activity was 5-fold faster. Thus, in vitro nicotianamine synthase activity depends highly on two additional enzymes, the inclusion of MTA-nucleosidase being vital. Although promising, cell-free nicotianamine production methods are not yet efficient enough for industry-scale efforts. Sequence and structural analyses suggest residues involved in azetidine ring formation and other aspects of the mechanism are explored.