Identification of a flavin‐containing S‐oxygenating monooxygenase involved in alliin biosynthesis in garlic

Summary S‐Alk(en)yl‐l‐cysteine sulfoxides are cysteine‐derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S‐alk‐(en)yl‐l‐cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin‐containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S‐oxygenation reaction in the biosynthesis of S‐allyl‐l‐cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S‐oxygenation of S‐allyl‐l‐cysteine to nearly exclusively yield (RCSS)‐S‐allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S‐oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin‐containing monooxygenases. AsFMO1 preferred S‐allyl‐l‐cysteine to γ‐glutamyl‐S‐allyl‐l‐cysteine as the S‐oxygenation substrate, suggesting that in garlic, the S‐oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre‐emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S‐allyl‐l‐cysteine S‐oxygenase, and contributes to the production of alliin both through the conversion of stored γ‐glutamyl‐S‐allyl‐l‐cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves. Significance Statement Garlic has metabolites, such as alliin, that are beneficial for human health. Here we characterize a flavin‐containing monooxygenase, AsFMO1, which catalyzes the S‐oxygenation reaction in alliin biosynthesis.