5hmf | pdb_00005hmf
Crystal structure of triazine hydrolase variant (P214T/Y215H/E241Q)
Structural highlights
Publication Abstract from PubMedThe 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 is known regarding how enzymes can naturally evolve active sites with highly reactive and desolvated charges. 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, Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a non-catalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214/His215). In order 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 raises the pKa of Glu241, enabling 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. Active site desolvation and thermostability tradeoffs in the evolution of catalytically diverse triazine hydrolases.,Sugrue E, Carr PD, Scott C, Jackson CJ Biochemistry. 2016 Oct 21. PMID:27768291[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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