Biochemical characterization of two Brassica oleracea nitrile-specifier proteins

<i>Brassica oleracea</i> vegetables (e. g. cabbages) form bioactive isothiocyanates (ITCs) from glucosinolate (GLS) hydrolysis. However, enzymatic activity, acidic pH (below pH 5), and ferrous ions (Fe²⁺) can promote nitrile release, reducing the ITC amount. In <i>Arabidopsis thaliana</i>, nitrile-specifier proteins (NSPs) promote nitrile formation upon GLS hydrolysis. Here, we report the functional characterization of two <i>Brassica</i> NSPs from <i>B. oleracea</i> and the <i>in silico</i> identification of candidate genes encoding a family of sixteen <i>B. oleracea</i> NSPs closely related to the <i>A. thaliana</i> NSPs and the likely ancestral protein, XP_013585314.1. High conservation of the iron-binding triad (EXXXDXXXH), characteristic of specifier proteins, was confirmed in the putative BoNSPs. Biochemical characterization of two <i>B. oleracea</i> NSP isoforms, BoNSP2 (XP_013609641.1) and BoNSP11 (XP_013587057.1), revealed increased NSP activity in the presence of added Fe²⁺. Both BoNSP isoforms affected hydrolysis of five GLS differently <i>in vitro</i>, suggesting differential substrate specificity. BoNSP2 showed higher nitrile formation from indol-3-ylmethyl GLS than from 4-(methylsulfinyl)butyl GLS. In contrast, BoNSP11 similarly increased nitrile formation from indol-3-ylmethyl GLS, three aliphatic GLS and benzyl GLS. BoNSP2 and BoNSP11 were most active between pH 7 and pH 8. This study identifies and characterizes the first NSPs in <i>B. oleracea</i> vegetables at the molecular level.