5EH9

Indirect contributions of mutations underlie optimization of new enzyme function

Summary for 5EH9

• Entry DOI: 10.2210/pdb5eh9/pdb
• Related: 3DHB
• Descriptor: N-acyl homoserine lactonase AiiA, ZINC ION, GLYCEROL, ... (5 entities in total)
• Functional Keywords: n-acyl homoserine lactonase from bacillus thuringiensis, hydrolase
• Biological source: Bacillus thuringiensis subsp. kurstaki
• Total number of polymer chains: 1
• Total formula weight: 29432.14

Authors

Hong, N.-S.,Jackson, C.J.,Tokuriki, N.,Yang, G.,Baier, F. (deposition date: 2015-10-28, release date: 2016-09-07, Last modification date: 2023-09-27)

Primary citation

Yang, G.,Hong, N.,Baier, F.,Jackson, C.J.,Tokuriki, N.
Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.
Biochemistry, 55:4583-4593, 2016

PubMed Abstract: 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 Å 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.

PubMed: 27444875
DOI: 10.1021/acs.biochem.6b00561

Experimental method

X-RAY DIFFRACTION (1.29 Å)