Biosynthetic enzyme complexes selectively catalyze challenging chemical transformations, including alkane functionalization (e.g., halogenation of threonine, Thr, by nonheme iron SyrB2). However, the role of complex formation in enabling reactivity and guiding selectivity is poorly understood, owing to the challenges associated with obtaining detailed structural information of the dynamically associating protein complexes. Combining over 10 µs of classical molecular dynamics of SyrB2 and the acyl carrier protein SyrB1 with large-scale QM/MM simulation, we investigate the substrate-protein and protein-protein dynamics that give rise to experimentally observed substrate positioning and reactivity trends. We confirm the presence of a hypothesized substrate-delivery channel in SyrB2 through free energy simulations that show channel opening with a low free energy barrier. We identify stabilizing interactions at the SyrB2/SyrB1 interface that are compatible with phosphopantatheine (PPant) delivery of substrate to SyrB2. By sampling metal-substrate distances observed in experimental spectroscopy of native SyrB2/SyrB1-PPant-S-Thr and non-native substrates, we characterize essential protein-substrate interactions that are responsible for substrate positioning, and thus, reactivity. We observe the hydroxyl sidechain and terminal amine of the native Thr substrate to form cooperative hydrogen bonds with a single N123 residue in SyrB2. In comparison, nonnative substrates that lack the hydroxyl interact more flexibly with the protein and therefore can orient closer to the Fe center, explaining their preferential hydroxylation and higher turnover frequencies.

Biosynthetic enzyme complexes selectively catalyze challenging chemical transformations, including alkane functionalization (e.g., halogenation of threonine, Thr, by non-heme iron SyrB2). However, the role of complex formation in enabling reactivity and guiding selectivity is poorly understood, owing to the challenges associated with obtaining detailed structural information of the dynamically associating protein complexes. Combining over 10 ms of classical molecular dynamics of SyrB2 and the acyl carrier protein SyrB1 with large-scale QM/MM simulation, we investigate the substrate–protein and protein–protein dynamics that give rise to experimentally observed substrate positioning and reactivity trends. We confirm the presence of a hypothesized substrate-delivery channel in SyrB2 through free energy simulations that show channel opening with a low free energy barrier. We identify stabilizing interactions at the SyrB2/SyrB1 interface that are compatible with phosphopantatheine (PPant) delivery of substrate to SyrB2. By sampling metal–substrate distances observed in experimental spectroscopy of native SyrB2/SyrB1-PPant-<i>S</i>-Thr and non-native substrates, we characterize essential protein–substrate interactions that are responsible for substrate positioning, and thus, reactivity. We observe the hydroxyl sidechain and terminal amine of the native Thr substrate to form cooperative hydrogen bonds with a single N123 residue in SyrB2. In comparison, non-native substrates that lack the hydroxyl interact more flexibly with the protein and therefore can orient closer to the Fe center, explaining their preferential hydroxylation and higher turnover frequencies.

Large scale quantum mechanical simulation systematically reveals length scales over which electronically driven interactions occur at enzyme active sites.

Nonheme iron halogenases, such as SyrB2, WelO5, and BesD, halogenate unactivated carbon atoms of diverse substrates at ambient conditions with exquisite selectivity seldom matched by nonbiological catalysts. Using experimentally guided molecular dynamics (MD) simulations augmented with multiscale (i.e., quantum mechanics/molecular mechanics) simulations of substrate-bound complexes of BesD and WelO5, we investigate substrate/active-site dynamics that enable selective halogenation. Our simulations reveal that active-site configurational isomerization is necessary in WelO5 to attain a substrate/active-site geometry consistent with its observed chemo- and regioselectivity. Conversely, a slight reorientation of the substrate from its crystal structure position is sufficient to enable regioselective chlorination in BesD without the need to invoke active-site isomerization. We observe very different patterns of substrate–protein interactions for these two enzymes, and we relate the nature of these interactions to the distinct substrates. For BesD, we resolve the uncertainty around the mechanistic relevance of Asn219. Our simulations reveal that the optimum substrate/active-site geometry also outweighs the interactions between the metal-oxo and the protein environment in facilitating the required chemoselectivity in halogenases. Our work highlights how different substrate-dependent strategies are used to accomplish selectivity-promoting proximity in halogenases.