Substrate Specificity of GSDA Revealed by Cocrystal Structures and Binding Studies

Area of Science:

• Biochemistry
• Structural Biology
• Plant Science

Background:

• Guanosine deaminase (GSDA) is crucial for nitrogen metabolism in plants, facilitating guanosine deamination.
• Previous studies elucidated Arabidopsis thaliana GSDA (AtGSDA) structures and identified novel substrates.
• The structural mechanisms governing AtGSDA activation and inhibition remain largely uncharacterized.

Purpose of the Study:

• To investigate the structural basis of AtGSDA inactivity towards certain guanosine derivatives.
• To elucidate the binding modes of ligands and their impact on enzyme activity.
• To understand the role of active site accessibility and ligand properties in GSDA function.

Main Methods:

• Cocrystallization of AtGSDA and its variants with various ligands, yielding 8 structures (1.85-2.60 Å resolution).
• Structural analysis of ligand-enzyme binding modes.
• Thermal shift analysis to assess enzyme stability and interactions.

Main Results:

• AtGSDA inactivity was linked to the absence of a 2-amino group in guanosine derivatives and the enzyme's inability to sequester its active site.
• Ligand binding was suboptimal due to poor interactions or unfavorable geometries at the active site.
• Conformational diversity was observed in the C-termini of AtGSDA under specific conditions.

Conclusions:

• Precise binding requirements and active site accessibility are critical for AtGSDA-mediated deamination.
• Ligand structural features significantly influence AtGSDA activity.
• These findings offer insights into the regulation and function of guanosine deaminase in plants.