Spectroscopically Guided Simulations Reveal Distinct Strategies for Positioning Substrates to Achieve Selectivity in Nonheme Fe(II)/α-Ketoglutarate-Dependent Halogenases

Nonheme iron halogenases, such as
SyrB2, WelO5, and BesD, halogenate
unactivated carbon atoms of diverse substrates at ambient conditions
with exquisite selectivity seldom matched by nonbiological catalysts.
Using experimentally guided molecular dynamics (MD) simulations augmented
with multiscale (i.e., quantum mechanics/molecular mechanics) simulations
of substrate-bound complexes of BesD and WelO5, we investigate substrate/active-site
dynamics that enable selective halogenation. Our simulations reveal
that active-site configurational isomerization is necessary in WelO5
to attain a substrate/active-site geometry consistent with its observed
chemo- and regioselectivity. Conversely, a slight reorientation of
the substrate from its crystal structure position is sufficient to
enable regioselective chlorination in BesD without the need to invoke
active-site isomerization. We observe very different patterns of substrate–protein
interactions for these two enzymes, and we relate the nature of these
interactions to the distinct substrates. For BesD, we resolve the
uncertainty around the mechanistic relevance of Asn219. Our simulations
reveal that the optimum substrate/active-site geometry also outweighs
the interactions between the metal-oxo and the protein environment
in facilitating the required chemoselectivity in halogenases. Our
work highlights how different substrate-dependent strategies are used
to accomplish selectivity-promoting proximity in halogenases.