Abstract
The Baeyer-Villiger monooxygenase (BVMO), LgnC, plays a crucial role in the biosynthesis of bacterial pyrrolizidine alkaloids, legonmycins. It processes bicyclic indolizidine substrates generated from the coordinative action of two non-ribosomal peptide synthetases (LgnB and LgnD) and the standalone type II thioesterase-like enzyme (LgnA). It has been demonstrated that the enzyme selectively inserts molecular oxygen into the carbon-carbon bond adjacent to the carbonyl group in legonindolizidines to form bicyclic 1,3-oxazepine carbamate intermediates. After ring opening and contraction, the most advanced products, prelegonmycins, are formed. However, factors controlling the final hydroxylation step and how the enzyme handles the substrates have remained elusive. In this study, we show that the final hydroxylation at the activated carbon of the electron-rich pyrrole system is attributed to either spontaneous oxidation or the action of an endogenous redox reagent. Substrate docking on the structural model of LgnC combined with site-directed mutagenesis allows the identification of several key amino acids that are essential for substrate/intermediate binding and a mechanism of LgnC-catalysed transformation is proposed.