Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana

Abstract

Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles.

Introduction

Glucosinolates (thioglucosides) are a class of sulphur containing secondary metabolites present in all cruciferous plants (Underhill, 1981). Glucosinolates and their degradation products are important bioactive compounds in the diet and health of animals and humans (Mithen, 2001). Myrosinase (E.C. 3.2.1.147) catalyses the hydrolysis of glucosinolates to give rise to an unstable thiohydroximate-O-sulphonate (Fig. 1), which can further rearrange to an isothiocyanate. Alternatively, in the presence of epithiospecifier protein (ESP) and Fe(II), epithionitriles are produced. ESP is unique in as much as it has no activity towards the initial substrate but only with the unstable thiohydroximate-O-sulphonate intermediate (Bones and Rossiter, 1996).

ESP has been independently purified to homogeneity (Bernardi et al., 2000, Foo et al., 2000) and has been shown for the first time to be a stable protein present in several isoforms (Foo et al., 2000). An anti-ESP antibody reacted with both the 39 and 35 kDa ESP forms in Brassica napus and also with a polypeptide corresponding to the 35 kDa ESP in Crambe abyssinica but is not present in all cruciferous plants (Foo et al., 2000). Partial amino acid sequencing of the 39 kDa ESP isoform revealed high similarity (81% identity) with a hypothetical 37 kDa protein from Arabidopsis thaliana (GenBank Accession Number H37255) and several jasmonate inducible proteins (Bernardi et al., 2000) while more recently ESP has been shown to be induced in the anthers of Arabidopsis by jasmonate (Mandaokar et al., 2003).

It has been suggested (Taipalensuu et al., 1996) that ESP is the previously identified myrosinase associated protein (MyAP), a glycoprotein, purified from B. napus. MyAP is wound and methyl jasmonate-inducible and has strong similarity to an early nodulin. However, we have recently shown that ESP does not require any specific myrosinase, as ESP is also active with myrosinase isolated from the aphid Brevicoryne brassicae (Foo et al., 2000, Jones et al., 2001).

A mechanism for the generation of epithionitriles via Fe(II) has been proposed (Foo et al., 2000). However, the Fe(II) dependency of ESP has recently been questioned (Lambrix et al., 2001). These authors expressed A. thaliana ESP in Eschericia coli and found no Fe(II) dependence. In A. thaliana ecotypes which do not express ESP a 10-bp deletion in the ESP promoter sequence was detected (Lambrix et al., 2001) and this deletion strongly correlates with a loss of ESP activity.

A. thaliana accession Col-5 has a glucosinolate profile similar to that of broccoli (Brassica oleracea var. italica) and contains glucoraphanin (4-methylsulfinylbutylglucosinolate) and on hydrolysis gives sulforaphane (1-isothiocyanato-4-(methylsufinyl)butane) as the major product. 1-Isothiocyanato-4-(methylsufinyl)butane has been shown to be an important anticarcinogen by inducing quinone reductase, a phase II detoxification enzyme in Hepa1c1c7 cell cultures (Zhang et al., 1992) and since this report many other studies supporting these findings have been published (Zhang et al., 1994, Gamet-Payrastre et al., 2000, Chiao et al., 2002, Fahey et al., 2002, Petri et al., 2003). Thus expression of ESP will be important in determining the bioactive profile of Brassica food crops.

We have set out to explore the possibility of using ESP to modify plant glucosinolate degradation profiles using Arabidopsis as a model. We use recombinant ESP to demonstrate an in vitro Fe(II) requirement for ESP activity in the formation of the thiirane ring.