An organic O donor for biological hydroxylation reactions

Abstract

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O₂, H₂O_2, or H₂O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O₂-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with ¹⁸O-shikimate, a precursor of prephenate, demonstrated the incorporation of ¹⁸O atoms into ubiquinone. The role of specific iron-sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments.

Keywords: U32 proteins; anaerobiosis; hydroxylation; prephenate; ubiquinone.

Fig. 1.

Aerobic and anaerobic pathways of UQ₈ biosynthesis in E. coli. The Ubi-enzymes common to both pathways are in black, those specific to the O₂-dependent pathway are shown in red, and those specific to the O₂-independent pathway are shown in blue (? for the unknown O- donor). The O atoms derived from the three hydroxylation steps are highlighted in yellow. R, octaprenyl chain shown in green in the UQ₈ structure; 4-HB, 4-hydroxybenzoic acid; OPP, 3-octaprenylphenol; UQ₈, ubiquinone-8.

Fig. 2.

O₂-independent biosynthesis of ubiquinone depends on the aro pathway in E. coli. (A) High Perfomance Liquid Chromatography-Electrochemical Detection (HPLC-ECD) analysis of lipid extracts from E. coli Δaro strains grown anaerobically in LB medium, UQ₁₀ added as internal standard. UQ₈ (B) and OPP (C) content in the strains analyzed in A. Mean ± SD (n = 3). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 by unpaired Student’s t test comparing to WT. ns, not significant. (D) Metabolic pathway of aromatic amino acids in E. coli, modified from ref. . The chorismate mutase (CM) and prephenate dehydratase (PDT) domains of PheA convert chorismate to prephenate and prephenate to phenylpyruvate, respectively (19). The black and gray arrows indicate the mutants not tested or tested in this study. The red and green disks indicate the proteins dispensable or essential for UQ₈ biosynthesis under anaerobic conditions, respectively. The ΔpheA and ΔtyrA strains are not deficient for UQ₈ but the double-mutant ΔpheA ΔtyrA is. The UQ pathway is highlighted in gray, and (D)MK₈ corresponds to the (demethyl) menaquinone pathway.

Fig. 3.

Prephenate is required for the O₂-independent biosynthesis of ubiquinone. The indicated E. coli strains were grown anaerobically in either LB (A and B) or MOPS (C and D) media. UQ₈ content in the mutants downstream of chorismate (A), in the ΔpheA ΔtyrA double mutant containing either pTet (vec), pTet-pheA (PheA), or pTet-pheA-CM [PheA (CM)] (B), in the double mutant ΔpheA ΔtyrA and the quadruple mutant ΔpheA ΔtyrA ΔubiIH strains supplemented or not with 1mM of prephenate (Preph) (C), in the ΔaroD and the ΔaroD ΔubiIH strains supplemented with 1 mM of prephenate and/or 10 µM 4-HB and/or 100 µM of shikimate (Shik) (D). Mean ± SD (n = 3) except for D (n = 2). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 by unpaired Student’s t test comparing to WT for (A), to vec for (B) and to mutant strains without supplementation for (C and D). (E) Serial dilutions of the mutant strains ΔubiIH, or ΔaroD ΔubiIH containing either pBAD24 (vec), pES077 (UbiIH), pES154 (UbiUV), or pES185 (UbiU(C176A)V) on M9 succinate plates containing Phe, Tyr, Trp, 0.02% arabinose and supplemented or not with 200 µM shikimate. (F) Serial dilutions of the ΔpheAΔtyrA ΔubiH strain containing either pTet + pEB067 (vec), pES047 + pEB067 (PheA_CM), pES047 + pES232 (PheA_CM + UbiUV), pTet + pES154 (UbiUV), or pES077 + pEB067 (UbiIH on M9 succinate or glucose plates containing Phe, Tyr and 0.1% arabinose. Incubation at 37 °C was carried out for 48 h under aerobic conditions for succinate plates (+O₂ Succ) or under anaerobic conditions for glucose plates (−O₂ Glc, used as controls) (E and F). The results are representative of at least two independent experiments (E and F).

Fig. 4.

Addition of C₄[¹⁸O]-shikimate to the ΔaroD strain leads to the biosynthesis of ¹⁸O₃-UQ in anaerobic conditions. (A) Synthetic route of ¹⁸O-shikimate (3R,4S,5R)-3,4[¹⁸O],5-trihydroxy-4-cyclohex-1-ene-1-carboxylic acid. i) APTS, CH₃OH; ii) TBDMSOtf, 2,6-lutidine, CH₂Cl₂; iii) Dess-Martin Periodinane, CH₂Cl₂, rt; iv) H₂¹⁸O, CH3C[¹⁸O]₂H, THF; v) a) NaBH₄, THF/H₂O; b) APTS, THF/H₂O. Mass spectra of UQ₈ from ΔaroD ΔubiIH strain grown anaerobically in the presence of 10 µM unlabeled ¹⁶O-shikimate (B) or C₄[¹⁸O]-shikimate (C). The peaks corresponding to adducts of either unlabeled UQ₈ or UQ₈ labeled with two ¹⁸O (¹⁸O_2-UQ₈) or three ¹⁸O (¹⁸O₃-UQ₈) are represented in blue (B) and red (C), respectively. Mass spectra representative of three independent experiments. (D) Quantification of UQ₈ H⁺ adducts (m/z 727 to 735) in WT and ΔaroD ΔubiIH cells grown with ¹⁶O-shikimate or C₄[¹⁸O]-shikimate. Mean ± SD (n = 3), see also SI Appendix, Fig S3.

Fig. 5.

Proposed molecular mechanism of prephenate-dependent hydroxylation: the reaction exemplifies the first hydroxylation step in the O₂-independent biosynthesis of UQ₈. R, octaprenyl chain.