Characterization and implications of Ca2+ binding to pectate lyase C

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Calcium ions (Ca2+) are essential cofactors for many enzymes, including pectate lyase C (PelC) from Erwinia chrysanthemi.
• Understanding the structural basis of Ca2+ interaction with PelC is key to elucidating its catalytic mechanism and function.

Purpose of the Study:

• To determine the structural and functional roles of Ca2+ in PelC activity across a range of pH conditions.
• To characterize the Ca2+ binding site and its influence on enzyme conformation and catalysis.

Main Methods:

• X-ray crystallography was used to solve the structures of 11 PelC-Ca2+ complexes at different pH values (4.5, 9.5, 11.2) and Ca2+ concentrations.
• pKa calculations were performed to assess the impact of Ca2+ on key amino acid residues.
• Enzyme activity assays were conducted at varying pH and Ca2+ concentrations.
• Intrinsic tryptophan fluorescence was used to estimate Ca2+ binding affinity.

Main Results:

• A novel Ca2+ binding motif involving beta-turns and beta-strands was identified in PelC.
• Ca2+ binding alters side-chain conformations of Asp-129 and Glu-166 and affects water molecule occupancy.
• Ca2+ binding lowers the pKa of Arg-218, a critical residue for catalysis.
• PelC exhibits weak Ca2+ affinity, with affinity varying significantly with pH.
• PelC activity is significantly enhanced at acidic pH (4.5) in the presence of saturating Ca2+.

Conclusions:

• The primary Ca2+ ion in PelC plays multiple roles, influencing both structure and catalytic activity.
• The pH-dependent Ca2+ affinity and activity suggest a complex regulatory mechanism for PelC.
• Structural insights into Ca2+ binding provide a foundation for understanding pectate lyase function and engineering.