Coq6 is responsible for the C4-deamination reaction in coenzyme Q biosynthesis in Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae is able to use para-aminobenzoic acid (pABA) in addition to 4-hydroxybenzoic acid as a precursor of coenzyme Q, a redox lipid essential to the function of the mitochondrial respiratory chain. The biosynthesis of coenzyme Q from pABA requires a deamination reaction at position C4 of the benzene ring to substitute the amino group with an hydroxyl group. We show here that the FAD-dependent monooxygenase Coq6, which is known to hydroxylate position C5, also deaminates position C4 in a reaction implicating molecular oxygen, as demonstrated with labeling experiments. We identify mutations in Coq6 that abrogate the C4-deamination activity, whereas preserving the C5-hydroxylation activity. Several results support that the deletion of Coq9 impacts Coq6, thus explaining the C4-deamination defect observed in Δcoq9 cells. The vast majority of flavin monooxygenases catalyze hydroxylation reactions on a single position of their substrate. Coq6 is thus a rare example of a flavin monooxygenase that is able to act on two different carbon atoms of its C4-aminated substrate, allowing its deamination and ultimately its conversion into coenzyme Q by the other proteins constituting the coenzyme Q biosynthetic pathway.

Keywords: Saccharomyces cerevisiae; coenzyme Q; deamination; flavin; hydroxylase; isotopic tracer; mass spectrometry (MS); monooxygenase; mutagenesis.

FIGURE 1.

S. cerevisiae Q₆ biosynthetic pathway. 4-HB and pABA differ by the presence of a hydroxyl (black) or an amino group (blue) at position C4. The numbering of the aromatic carbon atoms used on all Q₆ biosynthetic intermediates mentioned in this study is shown on the reduced form of Q₆, Q₆H₂. 4-HB and pABA serve as precursors for Q₆ biosynthesis and are prenylated by Coq2 to form HHB and HAB, respectively. R represents the hexaprenyl tail. The presence of a hydroxyl or an amino group at position C4 of intermediates is represented by OH/NH₂ and the respective names are indicated: DHHB, HHAB, DDMQ₆H₂, and DMQ₆H₂ are the reduced forms of demethyl-demethoxy-Q₆ (DDMQ₆) and demethoxy-Q₆ (DMQ₆); IDDMQ₆H₂ and IDMQ₆H₂ are the reduced forms of 4-imino-demethyl-demethoxy-Q₆ (IDDMQ₆) and 4-imino-demethoxy-Q₆ (IDMQ₆). The C4-deamination reaction occurs at an undefined step and IDMQ₆ is the most downstream amino-containing intermediate identified to date. Upon inactivation of coq6, HHB and HAB are decarboxylated (dashed arrow) and hydroxylated at position C1, yielding 4-HP₆ and 4-AP₆. Steps impaired in the Δcoq9 strain are designated with a red asterisk (*) for partial inactivation of the reaction, and double asterisk (**) for complete inactivation.

FIGURE 2.

Isotopic labeling of Q₆ in W303 cells. A, MS spectrum of Q₆ eluting at 10.4 min in the HPLC analysis of lipid extracts from W303 cells grown in YNB-p, 2% lactate (w/v), 2% glycerol (w/v) medium containing 50 μm pABA and 75% H₂¹⁸O (v/v). B and C, MS spectra of Q₆ from cells grown under ¹⁸O₂ atmosphere in YNB-p, 2% lactate, 2% glycerol medium containing 50 μm 4-HB (B) or 50 μm pABA (C).

FIGURE 3.

Conversion of 3H4AB into Q₆ requires an active Coq6. A, HPLC-ECD analysis of lipid extracts from 2 mg of WT (W303) cells grown in YNB-p, 2% galactose containing or not 10 μm 4-HB or 1 mm 3H4AB. The peaks corresponding to Q₆, DMQ₆, and to the internal standard Q₄ are marked. The chromatograms are shifted for better visualization but respect the scale of the y axis with the baseline corresponding to 0 μA. B, quantification of cellular Q₆ content (n = 5) of the same cells as in A in picomoles per mg of wet weight, error bars represent standard deviation. C, HPLC-MS analysis of Q₆ from WT cells grown under ¹⁸O₂ atmosphere in YNB-p, 2% lactate, 2% glycerol medium containing 1 mm 3H4AB. D, HPLC-ECD analysis of lipid extracts from 10 mg of Δcoq6 cells overexpressing COQ8 and grown in YNB-p, 2% galactose containing 10 μm 4-HB or 1 mm 3H4AB or 3,4-diHB. The peaks corresponding to DMQ₆, IDMQ₆, and 4-HP₆ are marked. E, HPLC-MS analysis of IDMQ₆ eluting at 11.6 min in the analysis of lipid extracts from Δcoq6 + pCOQ8 cells grown with 1 mm 3H4AB. F, HPLC-ECD analysis of lipid extracts from 10 mg of Δcoq6 cells expressing Coq6 G386A-N388D and grown in the same media as in D. The electrochromatograms are representative of 3 (D), 4 (F), or 5 (A) independent experiments.

FIGURE 4.

Point mutations in Coq6 affect the C4-deamination reaction. A, structural model of yeast Coq6⁶ as prepared with Discovery Studio Visualizer (Accelrys Software Inc.). Mutation sites are shown as spheres and the FAD is shown as sticks. The red corresponds to the 11 C-terminal residues, which are truncated in the M469X mutant. B, closer view of the active site (in a slightly different orientation compared with A) and of the residues important for deamination as prepared with PyMol. The substrate access tunnel is blue and C-terminal 11 residues are red. FAD, Leu-382, and G248R are shown as sticks and Arg-248 is superposed from the model of the G248R mutant.⁶ C, serial dilutions of Δcoq6 cells expressing WT Coq6 or the indicated mutants. The plates contained YNB-p, 2% glucose or 2% lactate, 2% glycerol and pABA or 4-HB at 20 μm as indicated. The plates were imaged after 3 (glucose) or 4 days (lactate/glycerol) at 30 °C. Results are representative of 3 independent experiments. D and E, HPLC-ECD analysis of lipid extracts from 1 mg of Δcoq6 cells expressing WT Coq6 or the designated Coq6 mutants and grown in YNB-p, 2% galactose containing 20 μm 4-HB or 20 μm pABA. The electrochromatograms are representative of 2 (D) and 3 (E) independent experiments.

FIGURE 5.

Genetic interaction between Coq6 and Coq9. A, HPLC-ECD analysis of lipid extracts from 4 mg of Δcoq6Δcoq9 + pCOQ8 cells expressing WT Coq6 or Coq6-M469X grown in YNB-p, 2% galactose containing 20 μm 4-HB or 20 μm pABA. The electrochromatograms are representative of 4 independent experiments. B, immunodetection of Coq6 in mitochondria prepared from Δcoq6 (lanes 1 and 2) and Δcoq6Δcoq9 + pCOQ8 cells (lanes 3 and 4) expressing WT Coq6 or Coq6-M469X. The mitochondrial proteins Anc2 and Por1 are used as loading control.

FIGURE 6.

Human Coq6 supports C4-deamination in yeast cells lacking Coq9. A, serial dilutions of Δcoq6 cells containing an empty vector (vec) or a centromeric vector expressing yeast Coq6 (yCoq6) or human Coq6 isoform 1 (huCoq6). The plates contained YNB-p glucose or lactate glycerol and pABA or 4-HB at 20 μm as indicated. The plates were imaged after 3 (glucose) or 10 days (lactate/glycerol) at 30 °C. Results are representative of 5 independent experiments. B, HPLC-ECD analysis of lipid extracts from 9 mg of Δcoq6 cells expressing human Coq6 grown in YNB-p, 2% galactose containing 20 μm 4-HB or 20 μm pABA. The electrochromatograms are representative of 4 independent experiments. C, HPLC-ECD analysis of lipid extracts from 5 mg of Δcoq6Δcoq9 + pCOQ8 cells expressing human Coq6 grown in YNB-p, 2% galactose containing 20 μm 4-HB or 20 μm pABA. The electrochromatograms are representative of 6 independent experiments. D, area of the electrochemical peaks (arbitrary units) corresponding to 4-AP₆, DMQ₆, and IDMQ₆ in lipid extracts from 7 mg of Δcoq6Δcoq9 + pCOQ8 + phuCoq6 cells grown in YNB-p 2% galactose containing 20 μm pABA or 20 μm ¹³C₇-pABA as indicated. The proportion of each compound, labeled (gray) or unlabeled (black), as determined by MS analysis is shown (n = 3), error bars represent S.D.

SCHEME 1.

Proposed mechanism for the C5-hydroxylation and C4-deamination reaction catalyzed by Coq6. Nucleophilic attack of HAB onto the flavin C4a-hydroperoxide (FAD-O-OH) results in the formation of the nonaromatic C5-hydroxylated product (step A). Rearomatization (step B) leads to HHAB. A second round of hydroxylation proceeds on carbon C4 according to a similar mechanism. Nucleophilic attack of HHAB onto FAD-O-OH results in the formation of the nonaromatic C4-hydroxylated product (step A′). Elimination of ammonia (step C′) facilitated by protonation of the amino group (step B′) leads to the formation of an intermediate o-quinone, which is then reduced into DHHB by 2 electrons and 2 protons. In Coq6 L382E or Coq6 M469X, hydroxylation at C4 is not efficient (see text for details) and the amino group on C4 is therefore not eliminated. R represents the hexaprenyl tail and the numbering of the carbon atoms is shown on HAB.