1 research outputs found
Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects
How remote mutations can lead to
changes in enzyme function at
a molecular level is a central question in evolutionary biochemistry
and biophysics. Here, we combine laboratory evolution with biochemical,
structural, genetic, and computational analysis to dissect the molecular
basis for the functional optimization of phosphotriesterase activity
in a bacterial lactonase (AiiA) from the metallo-β-lactamase
(MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase
activity is caused by a more favorable catalytic binding position
of the paraoxon substrate in the evolved enzyme that resulted from
conformational tinkering of the active site through peripheral mutations.
A nonmutated active site residue, Phe68, was displaced by ∼3
Ã… through the indirect effects of two second-shell trajectory
mutations, allowing molecular interactions between the residue and
paraoxon. Comparative mutational scanning, i.e., examining the effects
of alanine mutagenesis on different genetic backgrounds, revealed
significant changes in the functional roles of Phe68 and other nonmutated
active site residues caused by the indirect effects of trajectory
mutations. Our work provides a quantitative measurement of the impact
of second-shell mutations on the catalytic contributions of nonmutated
residues and unveils the underlying intramolecular network of strong
epistatic mutational relationships between active site residues and
more remote residues. Defining these long-range conformational and
functional epistatic relationships has allowed us to better understand
the subtle, but cumulatively significant, role of second- and third-shell
mutations in evolution