Gene isolation, heterologous expression, purification and functional confirmation of sesamin synthase from Sesamum indicum L.

Abstract

Members of Cytochromes P450 super family of enzymes catalyse important biochemical reactions in plants. Some of these reactions are so important that they contribute to enormous chemical diversity seen in plants. Many unique secondary metabolites formed by mediation of these enzymes play key role in plant defence and often contribute to maintenance of human health. In oilseed crop Sesamum indicum, the reaction leading to the formation of clinically important sesamin is catalyzed by a unique methylene-di-oxy bridge forming Cytochrome P450 enzyme sesamin synthase. It is encoded by the gene CYP81Q1. In order to elucidate the structure – function relationship of this enzyme and to apply biotechnological tools for enhancing the production of sesamin in the crop, it was intended to clone and express the enzyme in a heterologous system. In this paper we present our results on synthesis of cDNA, cloning, expression and purification of CYP81Q1 from the developing seeds of sesame crop. Following the same procedure we have also cloned a CYP reductase1 (CPR1) gene (CPR1) to facilitate transfer of electron from NADPH to CYP81Q1 enzyme from the same crop. Functional characterization was performed by expressing the recombinant proteins in E. coli (pET28a/BL21-DE3 codon plus) and its activity was evaluated in vitro by HPLC. We demonstrate that purified CYP81Q1 enzyme, on its own, has limited level of activity in the conversion of pinoresinol to sesamin. Its activity gets considerably enhanced in the presence of CPR1. Keywords: Sesamin, Cytochrome P450, Sesamin synthase, Heterologous expression, CYPR1