Abstract
Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron–electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.
Highlights
Several enzymes are known to have evolved from non-catalytic proteins such as solutebinding proteins (SBPs)
We investigated two plausible explanations for how remote mutations could have led to an increase in catalytic activity along this evolutionary trajectory: they may have (i) altered the sampling of rotamers of active site residues, such as the general acid Glu[173], thereby changing the structure and character of the active site and controlling the configuration of active site residues, and/or (ii) altered the equilibrium between open and closed states of the protein to minimise sampling of the catalytically unproductive open state
We previously showed that while AncCDT-1 binds L-arginine with comparable affinity to extant L-arginine-binding proteins, the P188 AncCDT-3 reconstruction, AncCDT-5 and PaCDT have all lost the ancestral ability to bind proteogenic amino acids[40]
Summary
Several enzymes are known to have evolved from non-catalytic proteins such as solutebinding proteins (SBPs). We performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron–electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity. Like ArgBP, CDT adopts a periplasmic type-II SBP fold, but instead of binding amino acids, it catalyzes the Grob-like fragmentation of prephenate and L-
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