Pyrrole heterocycles are widely used in biochemistry. For example, pyrroles are used to bind metal ions by porphyrins in primary metabolism. The metal binding capacity of pyrroles is exploited by bacterial secondary metabolites as well. In several secondary metabolites, pyrrole biosynthetic schemes are constructed via a type II Non-Ribosomal Peptide Synthetase (NRPS) adenylation-oxidation cascade which involves the 4e<sup>-</sup> oxidation of the pyrrolidine side chain of L-proline. Thio-templated pyrrole is a prevailing intermediate in the synthesis of numerous natural products where the pyrrole is tethered to a carrier protein (CP). We investigated the biocatalytic mechanism of proline-to-pyrrole synthesis using the recently reported structure of a Type II NRPS complex (Thapa et al., Biochemistry, 2019, 58, 918). The substrate (L-proline side chain) is attached to the CP Bmp1 and the catalytic site is located inside the flavin-dependent oxidase (Bmp3). After deprotonation of the C2 atom, the mechanism of biosynthesis requires oxidation of either the C2-C3 or the C2-N1 bond through hydride transfer either from C3 or N1. However, it is not yet known which atom donates the hydride to the FAD. Using molecular dynamics simulations, QM/MM simulations, and electronic structure calculations, we showed that the hydride transfer is more likely to occur from C3 than from N1 since it has a lower activation energy barrier. Furthermore, we showed that the FAD is stabilized in the catalytic site of Bmp3 by strong hydrogen bonding with Asn123 and Thr158.

Resolving the hydride transfer pathway in Bmp3-catalyzed pyrrole biosynthesis

ACS Fall 2021

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