Theoretical Investigation of the Functional Divergence of StnK3 via Site-Directed Mutagenesis: From Epimerase to Retro-Aldolase

StnK3 functions as an epimerase that catalyzes the chirality interconversion between (3<i>R</i>)- and (3<i>S</i>)-β-methylindolepyruvate (β-MeInPy). Strikingly, site-directed mutagenesis of His27 to Ala (H27A-StnK3) converts the wild-type (WT) enzyme from an epimerase into a retro-aldolase. A comprehensive understanding of the distinct catalytic mechanisms of both WT-StnK3 and the H27A mutant is critical to address the challenges in controlling stereochemical selectivity at the inert β-position and to elucidate the regulatory role of His27. In this study, based on molecular dynamics simulations and ONIOM calculations, we demonstrate that the epimerization of (3<i>R</i>)-β-MeInPy catalyzed by StnK3 proceeds through a substrate-assisted two-base mechanism. This mechanism involves a four-step proton transfer coupled with a one-step hydrogen bond rearrangement, distinguishing it from traditional one-base or two-base mechanisms. Furthermore, the H27A mutation induces functional alteration by facilitating the entry of water molecules into both <i>si</i>-face and <i>re</i>-face cavities of the substrate, leading to the formation of an organized water-mediated hydrogen-bond network. This network enables essential proton-transfer processes and mediates electrophilic α,β-addition. As a result, the structural reorganization caused by the incorporation of water molecules blocks the proton transfer from the substrate to Glu70, abrogating the native epimerization. Our results clarify the catalytic mechanisms of both wild-type StnK3 and the H27A variant, highlighting the significant role of His27 in modulating enzyme functionality. This research contributes new insights into the reaction mechanisms of epimerases and retro-aldolases, as well as the functional adaptability of multifunctional enzymes.