The Protein’s Role in Substrate Positioning and Reactivity for Biosynthetic Enzyme Complexes: The Case of SyrB2/SyrB1

Abstract

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.

Revealing Substrate Positioning Dynamics in Non-heme Fe(II)/αKG-dependent Halogenases Through Spectroscopically Guided Simulation

“…0.6 Å) to the iron-oxo moiety than typically observed for halogenases (e.g., SyrB2 34 ) and more distant (by ca. 1.7 Å) to Cl than would be expected 39 of a halogenase (Table 1). Moreover, the angle between the target substrate C-H bond and the ironoxo moiety is either too small (ca.…”

“…Crystal structures in cartoon representation and modeled iron-oxo intermediates in active sites for a) SyrB2-SyrB1 in complex with substrate Thr on the PPant arm 39 , b) BesD in complex with substrate Lys 14 , and c) WelO5 in complex with substrate 12-epi-fischerindole U 11 . The protein is shown in gray cartoon, and the substrate is shown in blue sticks or cartoon (i.e., for the SyrB1 carrier protein).…”

“…Additionally, DFT studies [28][29]33 evaluated the role of active-site configurational isomers in controlling halogenation vs. hydroxylation reactivity observed in SyrB2 6,35 . A spectroscopic study of SyrB2 34 invoked active-site isomerization to rationalize observations on substrate/active-site positioning, which we recently explored further using computational modeling 39 . Such studies are lacking for both BesD and WelO5 beyond the initial proposals made alongside published crystal structures.…”

“…Computational modeling of the dynamics of substrate-enzyme complexes can reveal the possible role of proteinsubstrate interactions in governing selective halogenation via strategic positioning. Additionally, simulations with otherwise experimentally elusive iron-oxo intermediates provide insight on catalytically operative active-site configurational isomers that are most aligned with experimentally-motivated (i.e., as guided by spectroscopic evidence) substrate positioning for halogenases [34][35]39 .…”

“…We recently used spectroscopically-derived 34 distance and angle metrics for substrate/active-site geometry to guide molecular dynamics (MD) simulations of halogenase SyrB2 with its substrates tethered to carrier protein. 39 This approach was necessary 39 because free molecular dynamics with approximate force fields can provide inaccurate descriptions of hydrogen bonding interactions and otherwise fail to adequately sample short, non-covalent distances. [40][41][42][43] Our simulations provided insight on previously unknown protein-substrate interactions that give rise to experimentally-measured substrate positions, which in turn impact reaction selectivity in SyrB2.…”