Resolving the Hydride Transfer Pathway in Oxidative Conversion of Proline to Pyrrole.
Biochemistry(2022)
Abstract
Thiotemplated pyrrole is a prevailing intermediate in the synthesis of numerous natural products where the pyrrole is tethered to a carrier protein (CP). Biosynthesis of the pyrrole requires oxidation of an L-proline side chain. Herein, we investigated the biocatalytic mechanism of proline-to-pyrrole synthesis using the recently reported (Thapa et al., Biochemistry, 2019, 58, 918) structure of a Type II non-ribosomal protein synthetase (NRPS) Bmp3-Bmp1 (Oxidase-CP) complex. The substrate (L-proline side chain) is attached to the Bmp1(CP) and the catalytic site is located inside the flavin-dependent oxidase (Bmp3). Interestingly, the FAD molecule is free (unbound) within the Bmp3 catalytic site. We show that the FAD isoalloxazine ring is stabilized in the catalytic site of Bmp3 by strong hydrogen bonding with Asn123, Ile125, Ser126, and Thr158. The stability of the dimeric Bmp3 system including one FAD molecule in our simulation suggests that the tetrameric Bmp3 assembly, as found in the crystal structure, is not functionally important. After the initial deprotonation followed by an enamine-imine tautomerization, oxidation of either the C2-C3 bond or the C2-N1 bond, through a hydride transfer (either from C3 or N1), is required for the pyrrole synthesis. Using molecular dynamics simulations, quantum mechanics/molecular mechanics simulations, and electronic structure calculations, we conclude that the hydride transfer is more likely to occur from C3 than N1. Additionally, we demonstrated the elasticity in the oxidase active site through enzymatic synthesis of proline derivatives.
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