Next, we assessed AfG3PS's control over the configuration at the glycerol unit. Previous work by the Poulter group and others firmly established that G3PSs are (S)-selective, although they all reported potential trace activity with (R)-GP and direct proof of the enantioselectivity has been missing from the literature. Mirroring this prior work, AfG3PS showed essentially no activity with (R)-GP (Fig. ), which is rationalized well by the crystal structure of AfG3PS reported by the Pai group (PDB ID 2f6x), showing complexation of (S)-GP via the highly conserved E167 (also see Fig. ). However, we did observe apparent trace activity with (R)-GP and obtained an additional crystal structure of His6-tagged AfG3PS in the presence of rac-GP (PDB ID 8ruw), which implies considerable flexibility of C2, C3 and the 3-OH group around the anchoring phosphate, as only the phosphate group of the ligand was well resolved (Fig. ). Thus, to rule out relevant activity with (R)-GP which would result in (R)-configured glycerol ethers, we used Kelly et al.'s diol derivatization method on dephosphorylated products of AfG3PS (Fig. , right). This yielded diastereomeric iminoboronates, which can be differentiated well by H NMR. Indeed, using rac-GP, all tested substrates were converted to purely (S)-configured products by AfG3PS, even when (R)-GP was supplied in excess (see 2G and please note the inversion of priority due to dephosphorylation, also see Fig. ). Although AfG3PS preferentially binds (S)-GP, the enantiomeric (R)-GP does bind weakly to the enzyme. As such, (R)-GP acts as a weak competitive inhibitor of (S)-GP binding and as a mixed inhibitor of binding of 3, 5 and 20 (Figs. and). A titration experiment monitored by 1 H NMR qualitatively confirmed this (Fig. ). The importance of the 2-OH group of GP is further underscored by the lack of binding and activity with 2-deoxy-GP (Fig. ).