Toward a Halophenol Dehalogenase from Iodotyrosine Deiodinase via Computational Design

Reductive
dehalogenation offers an attractive approach for removing
halogenated pollutants from the environment, and iodotyrosine deiodinase
(IYD) may contribute to this process after it can be engineered to
accept a broad range of substrates. The selectivity of IYD is controlled
in part by an active site loop of ∼26 amino acids. In the absence
of a substrate, the loop is disordered and only folds into a compact
helix-turn-helix upon halotyrosine association. The design algorithm
of Rosetta was applied to redesign this loop for response to 2-iodophenol
rather than iodotyrosine. One strategy using a restricted number of
substitutions for increasing the inherent stability of the helical
regions failed to generate variants with the desired properties. A
series of point mutations identified strong epistatic interactions
that impeded adaptation of IYD. This limitation was overcome by a
second strategy that placed no restrictions on side-chain substitution
by Rosetta. Nine representative designs containing between 14 and
18 substitutions over 26 contiguous sites were evaluated experimentally.
The top performing catalyst (UD08) supported a 4.5-fold increase in
turnover of 2-iodophenol and suppressed turnover of iodotyrosine by
2000-fold relative to the native enzyme. The active site loop of UD08
appeared less disordered than the native sequence in the absence of
substrate, as evident from their relative sensitivity to proteolysis.
Protection from proteolysis increased 9-fold for UD08 in the presence
of 2-iodophenol and nearly rivaled the equivalent response of wild-type
IYD to iodotyrosine. Thus, the Rosetta designs achieved the goal of
creating an active site sequence that gained structure in the presence
of iodophenol. Although a limited number of point mutations was sufficient
to increase the catalytic efficiency for 2-iodophenol dehalogenation,
only Rosetta successfully created a loop structure responsive to this
substrate.