Biochemical and structural explorations for [alpha]-hydroxyacid oxidases reveal a 4-electron oxidative decarboxylation reaction
Yeh HW, Lin KH, Lyu SY, Li YS, Huang CM, Wang YL, Shih HW, Hsu NS, Wu CJ, Li TL [1]
Molecular Tour
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)8-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 (FMNox) 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 FMNox 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, 18O-benzoate was detected, where the oxygen origin is proven from 18O2 rather than H218O2 confirming that free H2O2 is not the effective oxidant. 2) The level of H2O2 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.
- with a low average root-mean-square deviation (rmsd) of 0.064, where Hmo and Y128F are colored cyan and green, respectively.
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References
- ↑ Yeh HW, Lin KH, Lyu SY, Li YS, Huang CM, Wang YL, Shih HW, Hsu NS, Wu CJ, Li TL. Biochemical and structural explorations of alpha-hydroxyacid oxidases reveal a four-electron oxidative decarboxylation reaction. Acta Crystallogr D Struct Biol. 2019 Aug 1;75(Pt 8):733-742. doi:, 10.1107/S2059798319009574. Epub 2019 Jul 30. PMID:31373572 doi:http://dx.doi.org/10.1107/S2059798319009574
