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, As FMO 1, in garlic ( Allium sativum ), which is responsible for the S ‐oxygenation reaction in the biosynthesis of S ‐allyl‐ l ‐cysteine sulfoxide (alliin). Recombinant As FMO 1 protein catalyzed the stereoselective S ‐oxygenation of S ‐allyl‐ l ‐cysteine to nearly exclusively yield ( R C S S )‐ S ‐allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S ‐oxygenation reaction catalyzed by As FMO 1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin‐containing monooxygenases. As FMO 1 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 As FMO 1 is localized in the cytosol. As FMO 1 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 As FMO 1 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.