Next, we examined the substrate scope of AfG3PS, which yielded three principles describing the scope and selectivity of this promiscuous enzyme (Fig. ). Firstly, chain length governs affinity. In addition to 1, AfG3PS also converts the shorter substrates farnesyl (2, C15) and geranyl pyrophosphate (3, C10) with comparable rate constants. However, affinity decreases with chain length and -even across all accepted substrates and all profiled mutants (see below) -chain length alone is a good predictor of affinity (Fig. ). The shortest prenyl pyrophosphate (4, C5) is not converted, likely due to lack of binding. Secondly, AfG3PS preferentially converts substituted allylic pyrophosphates. While the analogues 5 and 6 (featuring a saturated tail) were converted, analogues 7 and 8 (featuring a saturated head motif) were not, which is in line with previous reports from the Poulter group on two related G3PSs which also failed to convert 7. Omitting the methyl group in 3-position (9 and 10) reduces activity to levels below the quantification limit of EPUB. However, AfG3PS does have trace activity with 10, which we confirmed by isolation of the corresponding ether product after prolonged incubation. We conclude that AfG3PS's lack of activity with substrates such as 7 or 9 is a problem of catalysis, not of binding, since binding studies by tryptophan fluorescence and with labelled enzyme confirmed that AfG3PS does bind these analogues (Fig. , the shorter analogues fail to interact sufficiently with the key fluorophore W99, Fig. ). As expected, fully saturated, linear alkyl pyrophosphates (11-17, C10-C18) are bound but not converted. Thirdly, when the conserved head group is retained, AfG3PS is tolerant of variations to the tail of the substrate. Supplementing the head group with simple alkyl The peaks labelled with an asterisk correspond to impurities or satellites and not the (2S)-boronate. b) Although (R)-GP is not converted as AfG3PS makes purely (S)-configured products, it does bind and act as a mixed inhibitor of the binding of pyrophosphates such as 3. c) AfG3PS's substrate preferences can be tuned by the introduction of mutations in the hydrophobic binding tunnel. Since the variants V80F and V80W did not display Michaelis-Menten-type kinetics with 5, their catalytic efficiencies are not available (n.a.). Please see the Supplementary Information for experimental details and the externally hosted supplementary material for all raw data. chains (18 and 19) or a phenyl group (20) gives competent substrates. Similarly, the 3-ethyl analogue 21 is converted, albeit more slowly. Although we performed these reactions in dilute, homogenous assay mixtures for the sake of kinetic analyses, these enzymatic transformations proved readily scalable and amenable to telescoped sequences with nonpurified pyrophosphates and slurry-to-slurry reactions (see the SI for details and examples).