Rieske nonheme iron oxygenases use two metallocenters,
a Rieske-type
[2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidation
reactions on a broad range of substrates. These enzymes are widely
used by microorganisms to degrade environmental pollutants and to
build complexity in a myriad of biosynthetic pathways that are industrially
interesting. However, despite the value of this chemistry, there is
a dearth of understanding regarding the structure–function
relationships in this enzyme class, which limits our ability to rationally
redesign, optimize, and ultimately exploit the chemistry of these
enzymes. Therefore, in this work, by leveraging a combination of available
structural information and state-of-the-art protein modeling tools,
we show that three “hotspot” regions can be targeted
to alter the site selectivity, substrate preference, and substrate
scope of the Rieske oxygenase p-toluenesulfonate
methyl monooxygenase (TsaM). Through mutation of six to 10 residues
distributed between three protein regions, TsaM was engineered to
behave as either vanillate monooxygenase (VanA) or dicamba monooxygenase
(DdmC). This engineering feat means that TsaM was rationally engineered
to catalyze an oxidation reaction at the meta and ortho positions of an aromatic substrate, rather than its
favored native para position, and that TsaM was redesigned
to perform chemistry on dicamba, a substrate that is not natively
accepted by the enzyme. This work thus contributes to unlocking our
understanding of structure–function relationships in the Rieske
oxygenase enzyme class and expands foundational principles for future
engineering of these metalloenzymes.
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