Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases

Abstract

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

Structural basis for UFM1 transfer from UBA5 to UFC1

“…The lack of a loop next to the UFC1 active site makes the latter heavily solvated by the electrophilic protons supplied by the water. This causes the active site Cys to be less potent for the nucleophilic attack, and thereby requires desolvation prior to its nucleophilic attack [33][34][35][36] . This motivated us to investigate whether UBA5 residues 347-377 play a role in UFC1 active site desolvation.…”

The properties of buried ion pairs are governed by the propensity of proteins to reorganize

“…Although dynamics have been invoked to explain the catalytic power of some enzymes 56575859 , the physical basis for how these motions are related to catalysis remains controversial 60 . Nevertheless, experimental work has suggested that evolution of effective enzymes requires a balance of stability and flexibility to effectively tune function 61 for specific environmental niches 62 . The results of this study suggest that conformational heterogeneity or flexibility can be used as a mechanism for tuning electrostatic energies for their functional roles.…”

“…The results of this study suggest that conformational heterogeneity or flexibility can be used as a mechanism for tuning electrostatic energies for their functional roles. Although the timescales of motions in this study are too slow to be relevant to catalysis, the relationship between flexibility 6361 and stability 62 , and the propensity of proteins to sample alternative states in response to charge burial, must be accounted for in the de novo design of novel enzymes.…”

Evolution of an enzyme from a solute-binding protein

“…Incorporation of the desolvated general acid Glu173 into the binding pocket of an ancestral SBP, despite initially being an adaptation for a different function, may have been sufficient for initial, promiscuous CDT activity. Indeed, the intrinsic reactivity of desolvated acidic and basic residues has been exploited similarly in enzymes that have evolved recently in response to anthropogenic substrates 20 and in enzymes engineered via single substitutions in non-catalytic proteins 21 . Following the introduction of a reactive general acid, optimization of enzyme-substrate complementarity and the introduction of hydrogen-bonding networks to position the catalytic residue precisely and stabilize the departing carboxylate group of the substrate appear to have occurred.…”