Journal:Acta Cryst D:S2059798319009574
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<b>Molecular Tour</b><br> | <b>Molecular Tour</b><br> | ||
The non-proteinogenic α-aminoacid, L-𝘱-OH-phenylglycine (L-𝘱-HPG), is a unique building unit found in several clinically important glycopeptide antibiotics, for example, vancomycin, teicoplanin and ramoplanin. Three enzymes (𝘚)-𝘱-OH-mandelate synthase (HmaS), (𝘚)-𝘱-OH-mandelate oxidase (Hmo) and L-𝘱-OH-phenylglycine transaminase (HpgT) are enzymes committed for biosynthesis of L-𝘱-HPG in three steps, in which Hmo is situated in the second step converting (𝘚)-𝘱-OH-mandelate to 𝘱-OH-benzoylformate. In terms of sequence similarity, Hmo is classified as a member of the flavin mononucleotide dependent oxidoreductase family. Hmo specifically acts on (𝘚)-mandelate but not its (𝘙)-mandelate enantiomer, while the α-hydroxyacid moiety is not limited to two carbon and the 𝘱𝘢𝘳𝘢/𝘮𝘦𝘵𝘢/𝘰𝘳𝘵𝘩𝘰 substituent on the phenyl ring has little impact on the enzyme reactivity. Beyond the biochemical analysis, how Hmo selects the substrates and executes the reaction remains largely unknown. We took advantage of X-ray crystallography in an attempt to snapshot Hmo/mutant crystals in complex with substrates, products, or inhibitors in a way to address these issues. | The non-proteinogenic α-aminoacid, L-𝘱-OH-phenylglycine (L-𝘱-HPG), is a unique building unit found in several clinically important glycopeptide antibiotics, for example, vancomycin, teicoplanin and ramoplanin. Three enzymes (𝘚)-𝘱-OH-mandelate synthase (HmaS), (𝘚)-𝘱-OH-mandelate oxidase (Hmo) and L-𝘱-OH-phenylglycine transaminase (HpgT) are enzymes committed for biosynthesis of L-𝘱-HPG in three steps, in which Hmo is situated in the second step converting (𝘚)-𝘱-OH-mandelate to 𝘱-OH-benzoylformate. In terms of sequence similarity, Hmo is classified as a member of the flavin mononucleotide dependent oxidoreductase family. Hmo specifically acts on (𝘚)-mandelate but not its (𝘙)-mandelate enantiomer, while the α-hydroxyacid moiety is not limited to two carbon and the 𝘱𝘢𝘳𝘢/𝘮𝘦𝘵𝘢/𝘰𝘳𝘵𝘩𝘰 substituent on the phenyl ring has little impact on the enzyme reactivity. Beyond the biochemical analysis, how Hmo selects the substrates and executes the reaction remains largely unknown. We took advantage of X-ray crystallography in an attempt to snapshot Hmo/mutant crystals in complex with substrates, products, or inhibitors in a way to address these issues. | ||
| - | 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 ( | + | 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 (FMN<sub>red</sub>) as the reductive half-reaction; FMN<sub>red</sub> 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, | + | 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, FMN<sub>red</sub> 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 FMN<sub>red</sub> 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. |
*<scene name='82/821049/Cv/3'>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. | *<scene name='82/821049/Cv/3'>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. | ||
Revision as of 12:52, 15 July 2019
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