The PD...(D/E)XK motif in restriction enzymes: a link between function and conformation.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Mg(II)-dependent nucleases possess conserved charged residues in their active sites, crucial for metal ion binding and catalysis.
• The PD-(D/E)XK motif in restriction enzymes highlights the importance of these residues.
• Previous studies suggested a link between active site charged groups and conformational behavior in nucleases.

Purpose of the Study:

• To investigate the relationship between active site residue alterations and conformational behavior in PvuII endonuclease.
• To analyze the impact of specific mutations on enzyme stability and structure-function dynamics.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy, including (19)F and (1)H-(15)N HSQC experiments, was used to probe structural changes.
• Thermodynamic methods were employed to assess conformational stability of wild-type and mutant enzymes.
• Analysis focused on site-directed variants at conserved active site residues Asp58, Glu68, and Lys70.

Main Results:

• NMR analysis revealed significant perturbations in side chain and backbone amide groups upon alanine substitution at active site residues.
• Mutations D58A and K70A, exhibiting the lowest activity, showed the most pronounced spectral changes.
• Mutant enzymes displayed increased conformational stability (2-5 kcal/mol) compared to wild-type, with distinct pH dependencies.

Conclusions:

• The conserved charged residues in Mg(II)-dependent nuclease active sites are critical for both catalytic function and conformational stability.
• Alterations in these residues significantly influence enzyme structure and dynamics, impacting DNA hydrolysis.
• Conformational stability measurements provide a valuable tool for evaluating site-directed mutant restriction enzymes and understanding complex structure-function relationships.