Intramolecular epistasis correlates with divergence of specificity in promiscuous and bifunctional <scp>NSAR</scp>/<scp>OSBS</scp> enzymes

Understanding the functions and evolution of specificity-determining residues is essential for improving strategies to predict and design enzyme functions. Whether the function of an amino acid residue is retained during evolution depends on intramolecular epistasis, which occurs when the same residue contributes to different phenotypes in different genetic backgrounds. This study examines the relationship between epistasis and functional divergence by investigating a conserved specificity determinant in five homologs from the N-succinylamino acid racemase (NSAR)/o-succinylbenzoate synthase (OSBS) subfamily. NSAR activity originated as a promiscuous (non-biological) activity of an ancestral OSBS. Some extant NSAR/OSBS subfamily enzymes still have OSBS activity as a biological function and NSAR as a promiscuous activity, while some use both OSBS and NSAR activities as biological functions. Others use only NSAR activity as a biological function but can still catalyze the OSBS reaction as a promiscuous activity. Previously, we determined that the conserved residue R266 in Amycolatopsis sp. T-1-60 NSAR contributes to NSAR specificity by enabling K263 to act as a general acid/base catalyst. Here, we show that mutating R266 decreased relative specificity for NSAR activity in four of five NSAR/OSBS subfamily enzymes, as predicted. However, other phenotypes exhibited epistasis related to the pleiotropy of R266, including the proton exchange rate between the catalytic lysines and the substrate, the impact on OSBS activity, and thermostability. The strength of epistasis was associated with functional and evolutionary divergence of NSAR/OSBS enzymes. These results illustrate the benefits of comparing multiple homologs for understanding mechanisms of enzyme specificity.