Substrate Tunnel Engineering Aided by X-ray Crystallography and Functional Dynamics Swaps the Function of MIO-Enzymes
Zsófia Bata, Zsófia Molnár, Erzsébet Madaras, Bence Molnár, E. W. Bell, Andrea Varga, Ibolya Leveles, Renzhe Qian, Friedrich Hammerschmidt, Csaba Paizs, Beáta G. Vértessy, László Poppe
Abstract
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.