Journal:Acta Cryst D:S2059798319009574
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The structure of Hmo is made of a single (α/β)<sub>8</sub>-barrel domain, in which an organic cofactor FMN serves as the prosthetic group with its redox-active isoalloxazine accessible to substrates or bulk solvents (for example, PDB entry [[5zzr]]). Based on the solved ternary complexes, six residues (F24, A79, Y128, M160, R163, H252, and R255) above the 𝘴𝘪-face of the isoalloxazine ring together position α-hydroxyacid in the substrate-binding site with α-H pointing toward N5 of isoalloxazine in a distance of 3.0 Å, agreeing with the reaction chirality (for example, PDB entries [[5zzr]] and [[6a08]]). Two active-site residues Y128 and H252 act as the catalytic dyad, where the distances and interactions between α-OH of α-hydroxyacid and Y128 (2.5 Å) or H252 (2.7 Å) support the direct-hydride transfer mechanism - H252 acts as the general base deprotonating α-OH to form an oxyanion that is stabilized by Y128. Upon collapse of the oxyanion, α-hydride is transferred to oxidized FMN (FMN<sub>ox</sub>) forming reduced FMN (FMNred) as the reductive half-reaction; FMNred then reacts with molecular oxygen forming a peroxide adduct prior to releasing as hydrogen peroxide in concomitance with the restoration of FMN<sub>ox</sub> as the oxidative half-reaction. | The structure of Hmo is made of a single (α/β)<sub>8</sub>-barrel domain, in which an organic cofactor FMN serves as the prosthetic group with its redox-active isoalloxazine accessible to substrates or bulk solvents (for example, PDB entry [[5zzr]]). Based on the solved ternary complexes, six residues (F24, A79, Y128, M160, R163, H252, and R255) above the 𝘴𝘪-face of the isoalloxazine ring together position α-hydroxyacid in the substrate-binding site with α-H pointing toward N5 of isoalloxazine in a distance of 3.0 Å, agreeing with the reaction chirality (for example, PDB entries [[5zzr]] and [[6a08]]). Two active-site residues Y128 and H252 act as the catalytic dyad, where the distances and interactions between α-OH of α-hydroxyacid and Y128 (2.5 Å) or H252 (2.7 Å) support the direct-hydride transfer mechanism - H252 acts as the general base deprotonating α-OH to form an oxyanion that is stabilized by Y128. Upon collapse of the oxyanion, α-hydride is transferred to oxidized FMN (FMN<sub>ox</sub>) forming reduced FMN (FMNred) as the reductive half-reaction; FMNred then reacts with molecular oxygen forming a peroxide adduct prior to releasing as hydrogen peroxide in concomitance with the restoration of FMN<sub>ox</sub> as the oxidative half-reaction. | ||
A single mutant Y128F turns itself an oxidase to a monooxygenase, whereby (𝘚)-mandelate is oxidized all the way to benzoate. Biochemical experiments were performed to conclude this finding: 1) In isotope labeling analysis, <sup>18</sup>O-benzoate was detected, where the oxygen origin is proven from <sup>18</sup>O<sub>2</sub> rather than H<sub>2</sub><sup>18</sup>O<sub>2</sub> confirming that free H<sub>2</sub>O<sub>2</sub> is not the effective oxidant. 2) The level of H<sub>2</sub>O<sub>2</sub> in the reactions with Y128 is inversely proportional to that with WT, indicating that the peroxide is a substrate in a well-organized manner with α-ketoacid, FMNred and active-site residues for the oxidative decarboxylation reaction to take place. 3) The structural complexes further reveal that reorientation of α-ketoacid from the 𝘱𝘳𝘰-S to a 𝘱𝘳𝘰-R configuration in Y128F makes FMNred or C4α-peroxide a nucleophile with a better attacking trajectory (for example, PDB [[6a19]]). As a result, the para-phenolic oxygen of Y128 in Hmo is determined to be a pivotal factor controlling the 2- or 4-electron oxidation reaction carried out by Hmo or Y128F, respectively. | A single mutant Y128F turns itself an oxidase to a monooxygenase, whereby (𝘚)-mandelate is oxidized all the way to benzoate. Biochemical experiments were performed to conclude this finding: 1) In isotope labeling analysis, <sup>18</sup>O-benzoate was detected, where the oxygen origin is proven from <sup>18</sup>O<sub>2</sub> rather than H<sub>2</sub><sup>18</sup>O<sub>2</sub> confirming that free H<sub>2</sub>O<sub>2</sub> is not the effective oxidant. 2) The level of H<sub>2</sub>O<sub>2</sub> in the reactions with Y128 is inversely proportional to that with WT, indicating that the peroxide is a substrate in a well-organized manner with α-ketoacid, FMNred and active-site residues for the oxidative decarboxylation reaction to take place. 3) The structural complexes further reveal that reorientation of α-ketoacid from the 𝘱𝘳𝘰-S to a 𝘱𝘳𝘰-R configuration in Y128F makes FMNred or C4α-peroxide a nucleophile with a better attacking trajectory (for example, PDB [[6a19]]). As a result, the para-phenolic oxygen of Y128 in Hmo is determined to be a pivotal factor controlling the 2- or 4-electron oxidation reaction carried out by Hmo or Y128F, respectively. | ||
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| + | *<scene name='82/821049/Cv/2'>Superposition of ternary complexes of wild type Hmo versus the Y128F mutant</scene> with a low average root-mean-square deviation (rmsd) of 0.064, where Hmo and Y128F are colored cyan and green, respectively. | ||
<b>References</b><br> | <b>References</b><br> | ||
Revision as of 10:16, 15 July 2019
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