Characterization of the Baeyer-Villiger monooxygenase in the pathway of the bacterial pyrrolizidine alkaloids, legonmycins

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.

Fig. 1. (A) Representative structures of naturally occurring bacterial pyrrolizidine alkaloids (PAs) and biosynthetically related lipocarbamates and cyclocarbamates. PAs 1–3 contain a hydroxyl group at the C7 position. (B) The biosynthesis of bacterial PAs and lipocarbamates only requires two essential enzymes, bimodular NRPS enzymes (C₁–A₁–T₁–C₂–A₂–T₂–Te) and diverged BVMOs, to provide these two distinct scaffolds. (C) A rather unusual pathway of legonmycins which rather require the presence of four enzymes, LgnA-D, to complete the biosynthesis. The atypical TEII enzyme domain, LgnA, is essential to catalyse the aminoacyl transfer between two T domains of two NRPSs, LgnB and LgnD.

Fig. 2. HPLC traces of the biotransformation from 8 to 3. (A) HPLC analysis of the conversion of 8 to 11 and 3 using purified recombinant LgnC-TF. Trace i. standard legonmycin A 3; ii. The standard 8; iii–vi. The time course assays showing the conversion of 8 to 11 and 3 (0 h, 2 h, 4 h, 6 h). (B) HPLC analysis of the conversion of 8 to 11 and 3 using cell-free extract of M1152-lgnC containing overexpressed recombinant His₆-LgnC. Trace i. the standard of 3; ii. The standard of 8; iii–vi. The time course assay shows the conversion of 8 to 11 and 3 (1 h, 2 h, 4 h, overnight).

Fig. 3. (A) HPLC analysis of the assays containing recombinant LgnC-TF with purified 11. Traces i-iii, standard compounds 11, 3, and 8, respectively. Trace iv. The negative control assay by incubating 11 with boiled LgnC-TF (inactivated). Trace v. The assay by incubating 11 with LgnC-TF. Trace vi. The positive control assay by incubating 8 with LgnC-TF. (B) HPLC analysis of the whole cell assays with either 8 or 11. Trace i, standard compound 11 with a trace amount of 3, possibly coming from spontaneous oxidation. Trace ii. The assay of incubating 11 with whole cells containing overexpressed LgnC-TF. Trace iii. The assay of incubating 11 with whole cells only containing empty plasmid. Trace iv. The assay of incubating 8 with whole cells containing overexpressed LgnC-TF. Trace v. The assay of incubating 8 with whole cells containing an empty plasmid.

Fig. 4. A model of LgnC active site bound with FAD cofactor (pink) was generated by AlphaFold 3. (A) Docked legonindolizidine 8 (salmon) interacting with key residues in the active site of LgnC. (B) Docked 1,3-oxazepine carbamate 7a (salmon) interacting with key residues in the active site of LgnC. (C) The relative enzyme activities of protein variants compared to wild-type LgnC. The biotransformation was measured from legonindolizidine A 8 to prelegonmycin A 11. (D) A plain structural elucidation of the interactions between LgnC key amino acid residues and legonindolizidine 8 (left) and 1,3-oxazepine carbamate 7a (right). Blue dashed lines represent hydrogen bonding interactions and brown dashed lines represent hydrophobic interactions.