Cyanuric acid hydrolase: evolutionary innovation by structural concatenation

Area of Science:

• Enzymology and Structural Biology
• Biochemistry
• Protein Structure and Function

Background:

• Cyanuric acid hydrolase (AtzD) is the first identified enzyme in a new class of ring-opening amidases.
• Understanding the structural basis of AtzD function is crucial for elucidating the catalytic mechanisms of this enzyme family.

Purpose of the Study:

• To determine the first X-ray structure of cyanuric acid hydrolase (AtzD) and characterize its novel fold.
• To identify the active site residues and elucidate the catalytic mechanism of AtzD.
• To investigate substrate specificity and evolutionary relationships within the AtzD enzyme family.

Main Methods:

• X-ray crystallography was employed to determine the structure of AtzD with bound cyanuric acid and inhibitors.
• Mutagenesis studies and biochemical evidence were used to identify active site residues.
• Homology modeling and phylogenetic analysis were performed to compare AtzD with related enzymes.

Main Results:

• The first X-ray structure of AtzD revealed a novel 'Toblerone' fold, likely formed by the concatenation of YjgF superfamily monomers.
• The active site was identified, featuring a threefold rotational symmetry with potential catalytic Ser-Lys dyads, with Ser85-Lys42 hypothesized as the active dyad.
• A plausible catalytic mechanism was proposed, and residues responsible for substrate specificity were inferred by comparing AtzD with barbiturase (Bar).

Conclusions:

• The 'Toblerone' fold represents a novel protein architecture for ring-opening amidases.
• The identified active site and proposed catalytic mechanism provide insights into the function of AtzD and related enzymes.
• Structural and phylogenetic analyses contribute to understanding the evolution and substrate specificity of the AtzD enzyme family.