Both aMD and IFD statistical models include features that describe how particular H172 conformations enhance selectivity, indicated by the negative sign associated with the coefficients. The aMD parameter corresponding to this residue measures the maximally extended conformation of H172 for each GluER-T36A variant. When H172 was extended, nearby residues N175 and Y177 were concomitantly displaced, resulting in the formation of a distinct binding pocket, shown as yellow spheres in both the cartoon and the aMD structure of GluER-T36A-F269L (Fig. ). Enzyme variants where H172 was retracted (Fig. , GluER-T36A-Y177W), resulted in occlusion of the putative binding pocket by nearby residues, leading to diminished enantioselectivity. We hypothesized an open binding site, as in GluER-T36A-F269L, resulted in facile substrate binding without significant steric rejection, which could induce substrate detrimental dissociation or rotation. These observations are also corroborated by the high conservation of H172 across 'ene'-reductase families, and studies on oxyanion holes in 'ene'-reductases. Figure . Mechanistic interpretation of descriptors in statistical models. a, Illustration of IFD and aMD ligand parameters from selected statistical models. Five-membered rings with aryl substituents typically had a more positive carbonyl oxygen NBO charge (greater than -0.73.) The NBO charge of the hydrogen attached to the product β-carbon was generally less than 0.2 for the products that resulted from 5-exo cyclizations (2a and 3a). The aMD steric descriptor (𝚫Sterimol Lsub) describes the flexibility of the substrate; it distinguished the most flexible substrate 4a (unscaled 𝚫Sterimol Lsub = 6.64 Å) from the less flexible substrates (1a = 0.14 Å, 2b = 1.19 Å). b, The Residue 172Sterimol L, max term from the aMD model indicated that extended configurations of H172 facilitated selectivity. Examination of enzyme conformers where this term was large (GluER-T36A-F269L = 6.7 Å) showed H172 to be extended (green) and revealed an open binding pocket (yellow sphere); this binding pocket was occluded in structures where values of this parameter were small (GluER-T36A-Y177W = 5.2 Å, purple) c, The aMD conformers demonstrate that when aromatic residues 100 and 177 were closely associated (green), interactions between residues 66 and 100 were precluded, which induced residue 66 flexibility and higher selectivity. The IFD conformational ensembles (right) corroborated that flexibility of residue 66 is necessary for substrate binding.