Lifetimes of the Aglycone Substrates of Specifier Proteins, the Autonomous Iron Enzymes That Dictate the Products of the Glucosinolate-Myrosinase Defense System in Brassica Plants

Abstract

Specifier proteins (SPs) are components of the glucosinolate-myrosinase defense system found in plants of the order Brassicales (brassicas). Glucosinolates (GLSs) comprise at least 150 known S-(β-D-glucopyranosyl)thiohydroximate-O-sulfonate compounds, each with a distinguishing side chain linked to the central carbon. Following tissue injury, the enzyme myrosinase (MYR) promiscuously hydrolyzes the common thioglycosidic linkage of GLSs to produce unstable aglycone intermediates, which can readily undergo a Lossen-like rearrangement to the corresponding organoisothiocyanates. The known SPs share a common protein architecture but redirect the breakdown of aglycones to different stable products: epithionitrile (ESP), nitrile (NSP), or thiocyanate (TFP). The different effects of these products on brassica consumers motivate efforts to understand the defense response in chemical detail. Experimental analysis of SP mechanisms is challenged by the instability of the aglycones and would be facilitated by knowledge of their lifetimes. We developed a spectrophotometric method that we used to monitor the rearrangement reactions of the MYR-generated aglycones from nine GLSs, discovering that their half-lives (t 1/2 ) vary by a factor of more than 50, from <3 to 150 s (22 °C). The t 1/2 of the sinigrin-derived allyl aglycone (34 s), which can form the epithionitrile product (1-cyano-2,3epithiopropane) in the presence of ESP, proved to be sufficient to enable spatial and temporal separation of the MYR and ESP reactions. The results confirm recent proposals that ESP is an autonomous iron-dependent enzyme that intercepts the unstable aglycone rather than a direct effector of MYR. Knowledge of aglycone lifetimes will enable elucidation of how the various SPs reroute aglycones to different products.

Ricca’s factors as mobile proteinaceous effectors of electrical signaling

“…The active membrane depolarization elicitors generated by TGG1 therefore have short half-lives. Thiohydroximate- O -sulfonates ( Figure 7 D), the aglycone breakdown products of GSLs, 26 were candidates for such molecules. A method to trap these unstable sulfur-rich intermediates in vitro has been developed: 2,2′-dipyridyl disulfide (2-PDS) traps GSL-derived aglycones in vitro without blocking myrosinase activity.…”

“…A method to trap these unstable sulfur-rich intermediates in vitro has been developed: 2,2′-dipyridyl disulfide (2-PDS) traps GSL-derived aglycones in vitro without blocking myrosinase activity. 26 Here, we employed 2-PDS for in vivo chemical trapping. To do this, WT plants were supplied with TGG1 alone or with TGG1 and 2-PDS.…”

Discovery, structure and mechanism of a tetraether lipid synthase

“…For reasons that we do not understand, the use of the pBAD42-BtuCEDFB plasmid greatly increased the solubility and yield of MJ0619. Although the plasmid was generated to enhance the solubility of cobalamin-containing proteins, it has also been found to enhance the solubility of some proteins that do not bind to cobalamin 46 . All remaining steps were performed in a Coy Laboratories anaerobic chamber or in an airtight vessel to ensure an oxygen-free environment.…”

Trendbericht Lebensmittelchemie

“…Kürzlich wurde erstmals die Halbwertszeit solcher Aglucone gemessen: Sie liegt bei 22 °C je nach Seitenkettenstruktur zwischen < 3 bis 150 Sekunden. 6) Die Abbauwege von Glucosinolaten in Kohlgemüsen beeinflussen nicht nur deren sensorische Qualität, sondern auch die Effekte auf die menschliche Gesundheit. Hierbei ist die Zubereitungsweise der Gemüse entscheidend.…”