Exploring the Substrate Switch Motif of Aromatic Ammonia Lyases

Aromatic ammonia lyases (AALs) are important enzymes for biocatalysis as they enable the asymmetric synthesis of chiral l-alpha-amino acids from the corresponding alpha,beta-unsaturated precursors. AALs have very similar protein structures and active site pockets but exhibit strict substrate specificity towards tyrosine, phenylalanine, or histidine. Herein, through systematic bioinformatics and structural analysis, we discovered eight new motifs of amino acid residues in AALs. After introducing them - as well as four already known motifs - into different AALs, we learned that altering the substrate specificity by engineering the substrate switch motif in phenylalanine ammonia lyases (PALs), phenylalanine/tyrosine ammonia lyases (PTALs), and tyrosine ammonia lyases (TALs) was only partially successful. However, we discovered that three previously unknown residue combinations introduced a substrate switch from tyrosine to phenylalanine in TAL, which was converted up to 20-fold better compared to the wild-type TAL enzyme. Using a bioinformatic approach, we identified novel substrate switch motifs of aromatic ammonia lyases. These alternative amino acids were introduced into a tyrosine ammonia lyase (TALrpc). The characterization of these enzyme variants revealed a significant (up to 20-fold) improvement in the activity for phenylalanine. A computational analysis could explain these experimental results.image

Automated summary

Researchers have discovered new substrate switch motifs in aromatic ammonia lyases and successfully altered the enzyme's substrate specificity. In particular, introducing previously unknown residue combinations enabled a switch from tyrosine to phenylalanine in tyrosine ammonia lyases.