Conformational changes and loose packing promote E. coli Tryptophanase cold lability

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Oligomeric enzymes can reversibly lose activity at low temperatures, a phenomenon known as cold lability.
• Tryptophanase (Trpase) from Escherichia coli, a pyridoxal phosphate (PLP)-dependent tetrameric enzyme, exhibits cold lability involving PLP release and dissociation.
• Understanding cold lability is crucial for protein stability and shelf-life applications.

Purpose of the Study:

• Investigate the mechanisms of reversible cold lability in Escherichia coli tryptophanase (Trpase).
• Examine the impact of specific mutations (Y74F, C298S, W330F) on Trpase cold lability and quaternary structure.
• Elucidate the role of PLP binding and hydrophobic interactions in Trpase stability.

Main Methods:

• Studied cold lability of wild-type and mutant E. coli Trpase at varying temperatures (2°C and 25°C).
• Determined crystal structures of Y74F and C298S apo-mutants at 1.9Å resolution.
• Utilized high-pressure studies to support conformational change observations.

Main Results:

• Apo forms of Trpase and its mutants dissociated into dimers at 25°C and further upon cooling to 2°C, unlike holo-Trpase.
• Crystal structures revealed apo mutants in an open conformation, contrasting with the closed conformation of holo P. vulgaris Trpase.
• High pressure studies corroborated the observed conformational changes.

Conclusions:

• PLP release is a primary driver of cold lability in E. coli Trpase.
• Mutations reducing amino acid side chain size enhance cold-induced activity loss by hindering tetramer assembly.
• Hydrophobic interactions at the non-catalytic interface significantly influence Trpase dissociation, explaining differences between E. coli and P. vulgaris Trpase behavior.