Author Archives: nYannik Brack, nChenghai Sun, nDong Yi, nUwe T. Bornscheuern

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

Posted on 15 March 2024 by nYannik Brack, nChenghai Sun, nDong Yi, nUwe T. Bornscheuern

Through a systematic analysis of protein sequences, several protein clusters of AAL-like enzymes with unusual MIO-forming motifs (ACG, TSG, SSG, and CSG) were identified, including two novel HALs with CSG motifs, one PAM with a TSG motif, and three novel ETLs without MIO motif. The systematic study and mutagenesis of MIO-groups in this work enable thus functional prediction and mutagenesis research of AALs.

Abstract

Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.

Exploring the Substrate Switch Motif of Aromatic Ammonia Lyases

Posted on 12 October 2023 by nYannik Brack, nChenghai Sun, nDong Yi, nUwe T. Bornscheuern

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 (TAL _rpc ). 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.

Abstract

Aromatic ammonia lyases (AALs) are important enzymes for biocatalysis as they enable the asymmetric synthesis of chiral l-α-amino acids from the corresponding α,β-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.