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

Biosynthetic
enzyme complexes selectively catalyze challenging
chemical transformations, including alkane functionalization (e.g.,
halogenation of threonine, Thr, by the non-heme iron halogenase 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 on 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 phosphopantetheine
(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 side chain
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.