Extended Data Fig. 7: Comparison of TokK structure with those of enzymes using alternative radical-generating mechanisms.

a–e, Residues that interact with the polar substituents in the β-lactam ring are shown in stick format for TokK (a) and the epimerase CarC (PDB ID: 4OJ8) (b). Although the two enzymes are structurally distinct, they use a similar number and type of functional groups to anchor the β-lactam by using direct and water-mediated contacts to the C7 carbonyl and C3 carboxylate substituents. In addition, comparison of the active site with those of hydroxylases reveals differences in the orientation of hydrogen atom transfer (HAT) intermediates and -OH or -CH₃ functionalization moieties. Fe(II)- and 2-oxo-glutarate-dependent (Fe-2OG) oxygenases use a ferryl [Fe(IV)-oxo)] intermediate to abstract an H-atom from an unactivated substrate carbon. The resulting Fe(III)-OH complex then couples with the substrate radical to yield a hydroxylated product. A vanadyl [V(IV)-oxo] mimic of the ferryl intermediate in L-Arg C3 hydroxylase, VioC, reveals that HAT and -OH transfer must occur from the same side of the substrate target carbon (c). The distance between the reactive oxo group and the substrate target carbon (indicated by the arrows) is 3.1 Å in VioC. A structure of the haem-dependent hydroxylase, P450cam, shows a similar phenomenon. A CO-bound mimic of the Fe(IV)-porphyrin radical intermediate, compound I, demands the same arrangement of HAT and -OH transfer components relative to the hydroxylation target on the camphor substrate (d). The distance between a mimic of the reactive group and the substrate target carbon (indicated by the arrows) is 3.1 Å in P450cam. By contrast, RS methylases use separate HAT reagents (5′-dA•) and methyl group donors (Me-Cbl), allowing for more diverse stereochemical outcomes in C-H functionalization reactions. (e) The structure of TokK in complex with carbapenam substrate, 1, shows a ~120° angle between the HAT acceptor (5′C of 5′-dAH), the substrate target carbon (C6), and the Cbl top ligand (-OH of OHCbl, a surrogate for Me-Cbl).