The base-promoted Kemp elimination reaction has been used as a model system for enzyme design. Among the multiple computationally designed and evolved Kemp eliminases generated along the years, the HG3-to-HG3.17 evolutionary trajectory is particularly interesting due to the high catalytic efficiency of HG3.17 and the debated role of glutamine 50 (Gln50) as potential oxyanion stabilizer. This study aims to elucidate the structural and dynamic changes along the evolutionary pathway from HG3 to HG3.17 that contribute to improved catalytic efficiency. In particular, we evaluate key variants along the HG3 evolutionary trajectory via molecular dynamics simulations coupled to non-covalent interactions and water analysis. Our computational study indicates that HG3.17 can adopt a catalytically competent conformation promoted by a water-mediated network of non-covalent interactions, in which aspartate 127 (Asp127) is properly positioned for proton abstraction and Gln50 and to some extent mutation cysteine 84 (Cys84) contribute to oxyanion stabilization. We find that HG3.17 exhibits a rather high flexibility of Gln50, which is regulated by the conformation adopted by the active site residue tryptophan 44 (Trp44). This interplay between Gln50 and Trp44 positioning induced by distal active site mutations affects the water-mediated network of non-covalent interactions, Gln50 preorganization, and water content of the active site pocket.
Keywords: Enzyme design; Kemp elimination; Molecular dynamics simulations; Non-covalent Interactions; Shortest path map.
© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.