Spectroscopically Guided Simulations Reveal Distinct Strategies for Positioning Substrates to Achieve Selectivity in Nonheme Fe(II)/α-Ketoglutarate-Dependent Halogenases

Abstract

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.

Mechanistic Insights Into Substrate Positioning Across Non-heme Fe(II)/alpha-ketoglutarate-dependent Halogenases and Hydroxylases

“…13 The prepared structure for WelO5 was obtained from ref. 29 . For all protein-substrate complexes, crystallizing agents were removed and missing residues were added using Modeller.…”

“…[22][23][24] Despite the intuitive role of the carboxylate substitution in halogenase reactivity, through the creation of a halide binding pocket, the re-introduction of a carboxylate through mutagenesis experiments does not confer native hydroxylase efficiency. 25 Further computational studies have revealed additional mechanisms that favor the challenging halogenation reaction over the thermodynamically preferred hydroxylation reaction such as direct positioning of the substrate, [26][27][28][29] active site isomerization, 30,31 inactivation of the hydroxyl ligand, 32,33 inherent reactivity of the rebound intermediate, 34,35 superior radical character on the halide, 36 and steric effects. 37 One of the most popular proposed mechanisms for selectivity control is the precise positioning of the substrate during halogenation.…”

“…38,39,42 Nevertheless, limitations in experimental methods to probe substrate positioning in the presence of the fleeting high-spin intermediate, especially among carrier-protein-dependent halogenases, have motivated key computational studies. [28][29][30][31] Notably, quantum mechanics/molecular mechanics (QM/MM) calculations and molecular dynamics (MD) simulations in conjunction with spectroscopically derived distance and angle positioning metrics, have revealed a key cooperative hydrogen bond between the carrier-proteindependent halogenase SyrB2 and its substrate L-threonine (L-Thr). 28 When similar spectroscopically-guided simulations were applied to the halogenases WelO5 43,44 and BesD, 45,46 they revealed that each enzyme utilizes distinct mechanistic strategies of maintaining an obtuse angle between the substrate, the metal center, and the oxo ligand.…”

“…28 When similar spectroscopically-guided simulations were applied to the halogenases WelO5 43,44 and BesD, 45,46 they revealed that each enzyme utilizes distinct mechanistic strategies of maintaining an obtuse angle between the substrate, the metal center, and the oxo ligand. [29][30][31] Obtuse substrate positioning angles have also been correlated to halogenase selectivity via the pand spathways, which favor halogenation and hydroxylation, respectively. 34 However, analogous spectroscopically-informed calculations have not been applied to hydroxylases, an important step in identifying mechanistic differences.…”

Regioselective control of biocatalytic C–H activation and halogenation

“…36,63 The selectivity of rebound has been hypothesized to be derived from precise control over the substrate radical positioning relative to the two potential rebounding species to allow for the more difficult halogenation reaction to occur. [43][44][45][46]64,65 . Although pairs of Fe II /αKG-dependent amino acid hydroxylases with regio-divergent outcomes have been reported 62,[66][67][68] , it is remarkable that the two halogenases, BesD and HalB, overcome the additional challenging of precisely orienting lysine within the active site to achieve regioselectivity without compromising selectivity for halogenation over hydroxylation.…”