Lastly, we sought to understand how AfG3PS orchestrates these transformations. For related G3PSs, a mechanism proceeding through a carbocationic intermediate has been proposed, based on the observation that 5 is converted and the corresponding fully saturated analogue (7) is not. This proposal mirrors known mechanisms of other prenyltransferases and the strict dependence of G3PS activity on divalent cations (Lewis acids like Mg 2+ , Zn 2+ or Mn 2+ ) supports that. Indeed, AfG3PS's substrate scope (Fig. ) and its Mg 2+ -dependence (Figs. 3b, S18, and S19) would also be consistent with such a mechanism. Yet how exactly this works has been an issue of speculation since i) reliable kinetic analyses of these enzymes and their mutants have historically been challenging and ii) no crystal structures featuring bound metals have been reported, neither for type I nor type II G3PSs (AfG3PS is a type I G3PS, please see the SI and Fig. for a discussion on their differences). To address this gap, we enlisted a combination of MD simulations with kinetic and biophysical characterization of mutants. We did attempt to obtain a crystal structure of AfG3PS with bound pyrophosphate substrate and/or bound Mg 2+ . However, despite extensive experimentation and the pronounced crystallinity of AfG3PS, we did not obtain a sufficiently resolved structure. Thus, we turned to MD simulations after identifying critical residues through alanine scanning and kinetic experiments with EPUB. Simulations with (E)-2 and (S)-GP suggested that both the pyrophosphate substrate and (S)-GP are heavily involved in Mg 2+ binding (Figs. and). We therefore hypothesize that, lacking a classical metal binding site, AfG3PS binds Mg 2+ only via the ensemble of organopyrophosphate and GP. Consistent with this hypothesis, AfG3PS has a remarkably low affinity for Mg 2+ (as assessed with kinetic experiments by continuous Please see the Supplementary Information for experimental details and the externally hosted supplementary material for all raw data. S18, and S19). In the absence of Mg 2+ , AfG3PS is fully capable of binding pyrophosphate substrates (as assessed by Trp fluorescence, Fig. ). Furthermore, all residues which our simulations implicated in Mg 2+ -binding are both highly conserved (Fig. ) and strictly required for activity (Figs. 3a and S29-S31 and Tables and). Some conservative modifications are permitted (e.g. the T39S, E167D and S169A/T mutants retain some residual activity and are capable of making enantiopure product from 3, Figs. and) and all active site mutations primarily affected rate. The other relevant properties, including affinity, solubility and stability, remained largely unchanged in all less active and inactive variants.