Discovery of a Cryptic Nitro Intermediate in the Biosynthesis of the 3-( trans -2'-Aminocyclopropyl)alanine Moiety of Belactosin A.

Belactosin A, a β-lactone proteasome inhibitor, contains a unique 3-( trans -2'-aminocyclopropyl)alanine moiety. We recently identified the biosynthetic gene cluster of the belactosin series from Streptomyces sp. UCK14. To shed light on the formation of the aminocyclopropylalanine, we established a heterologous pathway expression, constructed a set of gene deletion mutants, and performed feeding studies for a chemical complementation that include the incorporation of stable isotope-labeled precursors. We thereby show that, in the biosynthesis of this building block, a cryptic nitrocyclopropylalanine intermediate is generated from l-lysine. The subsequent reduction of the N-oxygenated precursor to the corresponding amine is mediated by the molybdopterin-dependent enzyme BelN.