Systematic Analysis of the MIO-forming Residues of Aromatic Ammonia Lyases

Area of Science:

• Biochemistry
• Enzymology
• Protein Science

Background:

• Aromatic ammonia lyases (AALs) and tyrosine/phenylphenylalanine ammonia mutases (TAM/PAM) are enzymes dependent on the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) moiety.
• The MIO moiety is typically autocatalytically formed from the Ala-Ser-Gly (ASG) tripeptide and functions as an electrophile in enzymatic reactions.

Purpose of the Study:

• To systematically investigate the diversity of MIO-forming residues within the AAL and TAM/PAM enzyme class.
• To identify novel MIO-forming residue variants and enzymes lacking the MIO motif.

Main Methods:

• In-depth sequence analyses of AAL-like enzymes.
• Identification of protein clusters with unusual MIO-forming residues (e.g., ACG, TSG, SSG, CSG).
• Site-directed mutagenesis of common MIO-forming groups to confirm the function of variants.

Main Results:

• Several clusters of AAL-like enzymes with non-canonical MIO-forming residues (ACG, TSG, SSG, CSG) were identified.
• Discovery of novel histidine ammonia lyases and one PAM with CSG and TSG residues.
• Identification of three novel ergothioneine trimethylammonia lyases that lack the MIO motif.
• Mutagenesis experiments confirmed the functionality of identified MIO variants.

Conclusions:

• The MIO-forming residues in AALs and TAM/PAMs exhibit greater diversity than previously recognized.
• These findings provide valuable insights for future functional predictions and targeted mutagenesis studies in this enzyme family.
• The discovery of MIO-less enzymes opens new avenues for research into alternative catalytic mechanisms.