Deciphering binding site conformational variability of substrate promiscuous and specialist enzymes

Substrate promiscuity is the ability of enzymes to catalyze reactions with alternate substrate(s) beyond their physiologically relevant ones. Such promiscuous activities expand enzymes functional landscape, enabling evolution or designing novel biochemical reactions. The molecular basis of substrate promiscuity remains elusive, though previous studies have suggested roles of active site structural features, such as flexibility, hydrophobicity, sub-sites, and electrostatics. Moreover, a recent hypothesis proposes the role of active site conformational variability in promiscuity. Accordingly, promiscuous enzymes could accommodate alternate substrate(s) through their pre-existing conformations, whereas specialists have a dominant conformation for their native substrates. To explore the role of active site conformational flexibility in substrate promiscuity, we compared the conformational states of two substrate promiscuous and specialist enzymes by analyzing their binding site structural dynamics from long-time explicit solvent molecular dynamics. Using tICA, we generated conformational states from simulations of the holo-conformation of enzymes. In addition to visual analysis of the variability of binding site conformation states, we performed quantitative estimation of the same using the native functionality score, which measures the contact similarity of conformations to the native structure. We observed that both specialist and generalist enzymes exhibited varied numbers of substrate binding site competent states, indicating that conformational flexibility to accept alternate substrates could exist in both groups of enzymes. Further, it caters to the view of substrate promiscuity as a continuum feature of enzymes.