Structural and Mechanistic Insights From High Resolution Crystal Structures of the Toluene-4-Monooxygenase Catalytic Effector Protein, NAD(P)H Oxidase and Choline Oxidase
Lountos, George Themistoclis
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X-ray crystallography provides detailed information of the atomic structure of macromolecules that aides in the understanding of their molecular function. In this study, the three-dimensional structures of the Toluene-4-monooxygenase catalytic effector protein (T4moD), NAD(P)H oxidase and choline oxidase were determined. The structures of wild-type and two mutant isoforms of T4moD were solved up to 1.7 resolution. Results from the crystallographic studies indicate that there are significant differences between the X-ray structure and the structure previously solved by NMR. The high-resolution structures have helped to define the potential differences in electrostatic surfaces that may govern the feasibility of protein-protein interactions and also reveal a single, well-defined cavity suitable for toluene binding that has substantial different electrostatic properties among the effector protein family members. The structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis was determined to 1.8 resolution. The flavoenzyme is of considerable interest as it catalyzes the oxidation of two equivalents of NAD(P)H and reduces one equivalent of oxygen to yield two equivalents of water without releasing hydrogen peroxide from the active site. The structure reveals the presence of a redox active cysteine residue that exists as a sulfenic acid and plays an important mechanistic role by reducing hydrogen peroxide to water. Additionally, a tightly bound ADP molecule was discovered in the enzyme which is hypothesized to play an important role in influencing the dual substrate specificity exhibited by the enzyme. The structure of choline oxidase from Arthrobacter globiformis was solved to 1.86 resolution. Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions. The structure reveals a cavity within the active site, which is suitable for choline binding. This allows for the identification of the putative binding site for choline and residues involved in substrate-binding and catalysis. Additionally, the structure reveals a highly distorted FAD cofactor that contains a C4a-adduct that is proposed to be either an FAD-C4a-OH or FAD-C4a-O2- complex.