In Vitro Characterization of Kitasetaline Biosynthesis Reveals a Bifunctional P450 Decarboxylase and a Vinyl β-Carboline Intermediate Susceptible to Nonenzymatic Thiol Addition

Kitasetaline is one of the very few β-carbolines isolated from bacteria. It features a unique <i>N</i>-acetylcysteine moiety linked to the β-carboline core through a thioether bond. While earlier <i>in vivo</i> experiments identified the gene cluster and reported several putative biosynthetic intermediates, how the C-S bond linkage is constructed has remained elusive. Herein, <i>in vitro</i> reconstitution of kitasetaline biosynthesis reveals the involvement of a Pictet-Spenglerase (KslB) and a promiscuous dehydrogenase (KslA) that generate the characteristic β-carboline ring system. In addition, the P450 enzyme KslC was found to catalyze oxidative decarboxylation of 1-(2-carboxyethyl)-9<i>H</i>-pyrido[3,4-<i>b</i>]indole-3-carboxylic acid to yield the biosynthetic intermediate 1-vinyl-9<i>H</i>-pyrido[3,4-<i>b</i>]indole-3-carboxylic acid. KslC is also capable of catalyzing further oxidation of its product to yield an <i>N</i>-hydroxylated side product. Importantly, the vinyl intermediate was found to undergo nonenzymatic nucleophilic addition by <i>N</i>-acetyl-l-cysteine to generate the C-S bond leading directly to kitasetaline without the involvement of a mycothiolated intermediate proposed in a previous biosynthetic model. Thus, this work not only demonstrates that biosynthesis of β-carboline compounds is rich in unexpected chemistry but also adds to the growing realization that biological thiolation reactions are often nonenzymatic in nature, relying instead on enzymatic formation of reactive electrophiles.