To assess the tunability of AfG3PS's inherent substrate preferences, we profiled a small set of AfG3PS mutants featuring altered sterics in their substrate binding "tunnel". In line with the high B-factors of this region, molecular dynamics (MD) simulations (using GROMACS 40 with the AMBER99SB-ILDN protein force field) with AfG3PS and bound substrates suggested considerable fluctuation of the covering loops to generate a rather exposed binding cleft (see the SI for details and Fig. ). We hypothesized that the large differences in rate constant between generally similar substrates (e.g. 3, 6 and 18) were at least in part due to steric interactions in the tunnel/cleft, which may be altered by mutagenesis. Indeed, mutating the highly conserved V80 located near the middle of the tunnel effected drastic changes in AfG3PS's substrate profile (Fig. ). Compared to the wild type, the sterically more crowded mutants V80F and V80W showed much higher catalytic efficiencies (up to tenfold; primarily due to increased rates) for 2, 18 and 21 and, surprisingly, did not show Michaelis-Menten-type kinetics with 5. This small sample size already provides proof of concept that the substrate scopes and preferences of G3PSs can be tuned by protein engineering. Although i) the other single mutants we fully profiled (H96W, Q98K, Q98N and W99A) showed only small changes in their substrate profile and ii) the rates with substrates lacking sufficient decoration of their tail (e.g. 18 and 19) still fell around two orders of magnitude short of the native substrate 1, we are confident that protein engineering of AfG3PS could yield highly active catalysts for other desired substrates.