We explore the role of substrate positioning in reaction selectivity exhibited by SyrB2, a representative mononuclear, non-heme Fe(II), alpha-ketoglutarate dependent biosynthetic enzyme. This class of non-heme Fe(II) enzymes have the ability to activate C-H bonds and catalyze a variety of reactions such as hydroxylation, halogenation and stereoinversion, making them crucial to the synthesis of valuable natural products. A deeper understanding of the factors that bias the enzymes towards a particular reaction is necessary for a deeper understanding of natural product biosynthesis and, ultimately, for enzyme engineering in therapeutic drug development. SyrB2 reacts only with substrates delivered via a prosthetic phosphopantetheine (PPant) arm delivered from a carrier protein. Depending on the loaded substrate, SyrB2 either exclusively chlorinates its native substrate L-Threonine or predominantly hydroxylates non-native substrates that differ only by absence of a hydroxyl group (L-Aminobutyric acid) as well as an added methylene group (L-Norvaline). The different reaction outcomes from subtle differences in substrates thus motivate us to identify how the greater SyrB2 protein environment biases selectivity for halogenation. Recent hyperfine sublevel correlation (HYSCORE) spectroscopy has provided some insight into average positions and angles of substrates to the iron center in SyrB2, but the approach cannot provide information about substrate-protein interactions that are likely responsible for those positioning trends. Here we use long-time (i.e., multi-microsecond) classical molecular dynamics (MD) and large-scale QM/MM to explain trends in HYSCORE substrate orientations and rationalize reaction specificity. By analyzing trends in hydrogen bonding, energy decomposition analysis, and large-scale electronic structure calculations, we study important protein-substrate and protein-protein interactions that possibly govern the dynamics of substrate positioning. The variance in the interactions sampled across the three substrates in three different coordination isomers allows us to: i) determine the unique interactions of the hydroxyl in L-Threonine that biases the enzyme to halogenation rather than hydroxylation for native substrate and ii) identify the most plausible coordination isomer of Fe(II) active site. Beyond selectivity in SyrB2, this approach is expected to provide insight into active-site dynamics in multi-protein biosynthetic assemblies.

Revealing substrate positioning dynamics in aliphatic halogenase SyrB2 through spectroscopically-guided simulation

255th National Meeting (2018)

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