ja5b02545_si_001.pdf (1.41 MB)
Mechanistic Investigation of the Radical S‑Adenosyl‑l‑methionine Enzyme DesII Using Fluorinated Analogues
journal contribution
posted on 2015-04-22, 00:00 authored by Geng-Min Lin, Sei-Hyun Choi, Mark W. Ruszczycky, Hung-wen LiuDesII is a radical S-adenosyl-l-methionine
(SAM) enzyme that can act as a deaminase or a dehydrogenase depending
on the nature of its TDP-sugar substrate. Previous work has implicated
a substrate-derived, C3-centered α-hydroxyalkyl radical as a
key intermediate during catalysis. Although deprotonation of the α-hydroxyalkyl
radical has been shown to be important for dehydrogenation, much less
is known regarding the course of the deamination reaction. To investigate
the role played by the C3 hydroxyl during deamination, 3-deutero-3-fluoro
analogues of both substrates were prepared and characterized with
DesII. In neither case was deamination or oxidation observed; however,
in both cases deuterium was efficiently exchanged between the substrate
analogues and SAM. These results imply that the C3 hydroxyl plays
a key role in both reactionsthereby arguing against a 1,2-migration
mechanism of deaminationand that homolysis of SAM concomitant
with H atom abstraction from the substrate is readily reversible when
forward partitioning is inhibited.