Structural investigation of MIO enzymes performing the conversion of aromatic amino acids have been selected as the topic of the project. A common feature of the enzyme family is the presence of 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO), a group of three amino acid residues (usually ASG), which is essential for catalysis. Among the MIO enzymes, the histidine, tyrosine and phenylalanine ammonia-lyases [HAL, TAL, PAL] eliminate ammonia from the corresponding amino acids, while the tyrosine and phenylalanine 2,3-amino mutases [TAM and (S)-PAM or (R)-PAM] catalyze the conversion of the corresponding α-amino acid into the β-amino acid.

Extending the scope of MIO enzymes to non-natural aromatic or even non-aromatic amino acids is to be based on structural studies at molecular level (protein crystallography, SAXS, and cryoEM) to facilitate structure-based enzyme modifications.

The promising preliminary results of the X-ray crystallography studies performed at BME ABFS 3Dstruct lab resulted in the solution of parsley PAL complexed with an inhibitor (see Figure) and the successful crystallization of several other MIO enzymes. Based on these resuts, the project aims to further elucidate the mechanism of action of MIO enzymes to facilitate structure-guided tailored genetic modifications. The changes may facilitate the extension of the substrate scope to other substrates that are significantly different from the natural substrate, or even accept D-α amino acids as substrates instead of the natural L-α amino acids.

The changes in size and shape of large MIO enzymes can be investigated in solution phase under variable conditions (e.g. temperature, pH, ionic strength, etc.) by small angle X-ray scattering (SAXS). Thus, it makes possible to follow the conformation changes associated with alterations of the enzyme activity.

In the course of the work, we plan to recombinantly produce four MIO enzyme families [PAL, HAL, (S)-PAM, and (R)-PAM] and other MIO enzymes of interest later (gene synthesis, cloning, expression optimization, recombinant expression); their crystallization and solution phase experiments with inhibitors or natural substrates; X-ray crystallographic, SAXS and cryoEM investigations of the complexes and the investigation of the mechanisms using computer simulations (MD, QM / MM calculations) aided by structural data.

The project team is made up of a number of young scientists alongside the seniors, so beyond their direct results, it is important for them to acquire internationally recognized research knowledge. The research is linked to several ongoing projects (NKFI SNN, CELSA, NeMSyB).

László Poppe, Attila Bóta, Beáta Vértessy

Result_May 2020