We therefore propose that catalysis happens via a pseudo-carbocationic intermediate featuring a stretched C-O bond via Lewis acid catalysis by Mg 2+ . Given the retention of alkene configuration and the lack of futile hydrolysis in the immensely solvent-exposed active site of the enzyme (Fig. ), we hypothesize that no free carbocation (such as is present in terpene synthases) is involved. Rather, a shortlived pseudo-cation is selectively intercepted by attack of (S)-GP's 3-OH, a process facilitated by its increased acidity via the action of a duo of catalytic bases, K11 and E64 (which likely plays a dual role during catalysis, Fig. ). This mechanism is enabled by a precise placement of catalytic residues. Both the pseudo-cation and the two Mg 2+ centers are stabilized by an arrangement of negative charges and Hbonding partners in D13, T39 and E64 (for Mg1), and E167 and S169 (for Mg2, Fig. ). The residues E64, K11 and E167 are locked tightly in place by an elaborate H-bonding network and positioning on rigid backbone structures (as indicated by their B-factors and confirmed by MD simulations, Fig. ). (S)-GP also engages in H-bonding with K11 and E167 (which also plays a dual role) to position them for Mg 2+ binding. The loop bearing D13 is positioned and held in place (Fig. ) by a trio of amino acids engaged in a separate H-bonding network adjacent to the active site (R16, N216, E220, Fig. ). Any disruption to this network yields an (almost) inactive enzyme, which is still capable of binding substrates but fails to effect efficient catalysis (Figs. and). Collectively, this mechanistic proposal rationalizes, i) the lack of bound Mg 2+ in all available crystal structures of G3PSs and ii) the strict requirement for the presence and (S)configuration of GP's 2-OH. MD simulations also suggested that (Z)-configured substrates can be engaged in a very similar catalytic ensemble thanks to the exposed binding cleft (Fig. ), rationalizing the observed conversion of (Z)pyrophosphates to (Z)-ethers.