Biosynthetic origin of the nitrogen atom in cyanamide in Vicia villosa subsp. varia

Natural cyanamide (NH₂CN) has recently been found in three Leguminosae plants: Vicia villosa subsp. varia, Vicia cracca and Robinia pseudo-acacia. As cyanamide has long been thought to be absent in nature, its physiological role and biosynthesis are totally unknown. In the present study, we demonstrated the incorporation of ¹⁵N from [¹⁵N]nitrate and [¹⁵N]ammonium into cyanamide using shoots of V. villosa subsp. varia, which ruled out the possibility that nodules are essential in cyanamide biosynthesis. We also applied [¹⁵N₂]cyanamide to shoots of V. villosa subsp. varia to monitor its turnover, and detected [ ¹⁵N₂]cyanamide in the leaves within 4 h; it was present without detectable degradation for more than 4 days. In contrast, maximum incorporation of ¹⁵N into cyanamide molecules was observed after 4 days of feeding the shoots with ¹⁵N-labeled inorganic ions and l-[amide-¹⁵N]-glutamine, indicating that these nitrogenous compounds are distant precursors of cyanamide. Although the guanidino group of l-arginine (-NH-C(NH₂)=NH) and urea (NH₂C(=O)NH₂) were candidate precursors of cyanamide on the basis of structural similarity, direct incorporation of the guanidino group of l-[¹³C₆, ¹⁵N₄]-arginine and [¹³C,¹⁵N ₂]urea into cyanamide was not observed. These results eliminated the possibility that cyanamide is biosynthesized by the addition of ammonia to an electrophilic carbon or by the conversion of the tested compounds that were structurally relevant to cyanamide.