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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.

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Introduction Cigarette smoke contains highly reactive free radicals thought to play an important role in tobacco smoke-induced harm. Previously, large variations in free radical and toxicant output have been observed in commercial cigarettes. These variations are likely due to cigarette design features (paper, filter, additives), tobacco variety (burley, bright, oriental, etc.), and tobacco curing methods (air, sun, flue, fire). Previous reports show that tobacco varieties and curing methods influence the production of tobacco smoke constituents like the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK). Methods We evaluated free radical, nicotine, and NNK production in cigarette smoke from cigarettes produced with 15 different types of tobacco. Gas-phase free radicals were captured by spin trapping with N-tert-butyl-α-phenylnitrone (PBN) and particulate-phase radicals were captured on a Cambridge Filter pad (CFP). Both types of radicals were analyzed using electron paramagnetic resonance (EPR) spectroscopy. Nicotine and NNK were extracted from the CFP and analyzed by gas chromatography flame ionization detection (GC-FID) and liquid chromatography mass spectrometry (LC-MS), respectively. Results Gas-phase radicals varied nearly 8-fold among tobacco types with Saint James Perique tobacco producing the highest levels (42 ± 7 nmol/g) and Canadian Virginia tobacco producing the lowest levels (5 ± 2 nmol/g). Nicotine and NNK levels in smoke varied 14-fold and 192-fold, respectively, by type. Gas-phase free radicals were highly correlated with NNK levels (r = 0.92, p &lt; 0.0001) and appeared to be most impacted by tobacco curing method. Conclusions Altogether, these data suggest that tobacco types used in cigarette production may serve as a target for regulation to reduce harm from cigarette smoking. IMPLICATIONS Variations in cigarette free radical and NNK levels vary based on the tobacco variety and curing method. Reducing the ratio of high producing free radical and NNK tobacco types offer a potential tool for regulators and producers looking to reduce toxicant output from cigarettes.