Substrate Tunnel Engineering Aided by X‑ray Crystallography and Functional Dynamics Swaps the Function of MIO-Enzymes

• Published in: ACS catalysis Vol. 11; no. 8; pp. 4538 - 4549
• Main Authors: Bata, Zsófia, Molnár, Zsófia, Madaras, Erzsébet, Molnár, Bence, Sánta-Bell, Evelin, Varga, Andrea, Leveles, Ibolya, Qian, Renzhe, Hammerschmidt, Friedrich, Paizs, Csaba, Vértessy, Beáta G, Poppe, László
• Format: Journal Article
• Language: English
• Published: American Chemical Society 16.04.2021
• Subjects: phenylalanine 2,3-aminomutase; mechanism-based inhibition; class I lyase-like enzyme; substrate-binding dynamics; crystal structure; phenylalanine ammonia-lyase; tunnel engineering; biocatalysis
• ISSN: 2155-5435; 2155-5435
• DOI: 10.1021/acscatal.1c00266

The enzyme family harboring the post-translationally formed 5-methylene-3,5-dihydro-4H-imidazol-4-one (MIO) catalytic residue comprises both aromatic amino acid ammonia-lyases (ALs) and 2,3-aminomutases (AMs). The structural origin of the different functions and the role of the inner loop region in substrate binding are not fully understood. Here, we provide the three-dimensional structures for Petroselinum crispum phenylalanine AL (PcPAL) with fully resolved inner loops in a catalytically competent conformation. Using molecular modeling, we demonstrate that in both ALs and AMs of eukaryotic origin, just a small opening of the inner loop is sufficient for ligand egress. Furthermore, we show that ligand-binding tunnels are analogous to eukaryotic MIO-enzymes and that the critical initial part of these tunnels is present across the whole enzyme family. Engineering of these binding tunnels converts an (R)-AM to a highly selective (S)-β-AL thus establishing a nonclassified enzyme function.