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Covering: 2015 to 2024Mass spectrometry (MS)-based methods have been implemented extensively for enzyme engineering due to their label-free nature, making them suitable for screening a wide range of b...

Tambjamines are complex bipyrrole-containing natural products that possess promising bioactive properties. Although Pseudoalteromonas citrea is known to produce both cyclic tambjamine MYP1 and the linear precursor (YP1), the biosynthetic machinery used to catalyze the site-selective oxidative carbocyclization at the unactivated 1° carbon of YP1 has remained unclear. Here, we demonstrate that a three-component Rieske system consisting of an oxygenase (TamC) and two redox partner proteins is responsible for this unprecedented activity on YP1 and potentially, a non-native substrate (BE-18591). We also show that a homologous oxidase from Pseudoalteromonas tunicata (PtTamC) can function together with the partner proteins from P. citrea to process both YP1 and BE-18591. These reactions represent the first Rieske oxygenase-catalyzed activations of C–H bonds at 1° carbons, resulting in carbon–carbon bond formation. The use of TamC and PtTamC to potentially generate the new-to-nature cyclic analogue of BE-18591 suggests the enormous biocatalytic potential of these Rieske systems to facilitate late-stage oxidative cyclizations at terminal C(sp3)–H bonds.