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![[Figure 3]](ag5031fig3_combined_mag.jpg)
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Figure 3
The effect of 4-OH of Tyr128 on polarization, hydrogen-bond networking and reactivity. (a, b) A close-up view of the wedge-shaped electron density on top of C4α=N5 of FMNox shown by unbiased difference electron-density maps (blue, positive electron density) contoured at 4σ in the wild type (a) and the Y128F mutant (b), suggesting that the electrons in the π-orbital of C4α=N5 are polarized from C4α to N5 (to form a C4α+–N5− ylide), with this being more significant in the Y128F mutant than in the wild type. (c, d) The point mutation Y128F disturbs the active-site hydrogen-bonding network between water, FMN and the catalytic dyad [the Y128F mutant loses the hydrogen bond between Tyr128 and H2O (187) but gains a new hydrogen bond between His252 and H2O (257)]. (e) Hydrogen peroxide was modeled at C4α, where the distance between Tyr128 and the terminal O atom of Cα-OOH is 2.7 Å. (f, g) The phenyl ring of benzoylformate, which is bulkier and takes up space, limits the access of dioxygen to the reaction center, while the methyl group of pyruvate, which is smaller and takes up less space, allows the access of dioxygen to the reaction center [the phenyl ring that bulges out at the substrate entrance in (f) prohibits exposure of FMNred to the bulk solvent, as opposed to the methyl group in (g) which allows exposure of FMNred to the bulk solvent]. Therefore, the size of the substrates is another factor in leverage of the oxidation cascade. (h) Aside from the active-site perturbation effect, the absence of the p-OH group also introduces some space allowing access of O2 to the C4α redox-active center. (i) The sulfhydryl group (SH) of the Y128C mutant has been oxidized to a sulfenyl group (S-OH), as it is vulnerable to ROS generated in the active site. Free ligands, FMN and active-site residues are colored cyan, yellow and green, respectively. |
![[Figure 3]](ag5031fig3_combined.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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