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Coq6 Is Responsible for the C4-deamination Reaction in Coenzyme Q Biosynthesis in Saccharomyces cerevisiae*

  • Mohammad Ozeir
    Footnotes
    Affiliations
    University of Grenoble Alpes, LCBM, UMR5249, F-38000 Grenoble, France
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  • Ludovic Pelosi
    Affiliations
    University of Grenoble Alpes, LAPM, F-38000 Grenoble, France

    CNRS, LAPM, F-38000 Grenoble, France
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  • Alexandre Ismail
    Affiliations
    Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, UPMC, Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France

    Sup'Biotech, IONIS Education Group, 66 rue Guy-Moquet, F-94800 Villejuif, France
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  • Caroline Mellot-Draznieks
    Footnotes
    Affiliations
    Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, UPMC, Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France
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  • Marc Fontecave
    Footnotes
    Affiliations
    Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, UPMC, Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France
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  • Fabien Pierrel
    Correspondence
    To whom correspondence should be addressed: Laboratoire Adaptation et Pathogénie des Microorganismes, UMR5163 UJF-CNRS, Université Grenoble Alpes, Institut Jean-Roget, Domaine de la Merci, 38700 la Tronche, France. Tel.: 33-4-76-63-74-79.
    Affiliations
    University of Grenoble Alpes, LAPM, F-38000 Grenoble, France

    CNRS, LAPM, F-38000 Grenoble, France
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  • Author Footnotes
    * This work was supported in part by the Région Rhône-Alpes program CIBLE 2009 and ANR pABACoQ (to F. P.) and French State Program “Investissements d'Avenir” Grants LABEX DYNAMO and ANR-11-LABX-0011 (to M. F. and C. M. D.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Present address: Laboratoire de Pharmaco-toxicologie et Biologie Structurale, INSERM UMR-S-1124, Univ. Paris Descartes, 45 rue des Saints-Pères, 75270 PARIS Cedex 06, France.
    2 Supported by the Fondation de l'Orangerie.
    3 Supported by the Fondation de l'Orangerie.
      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.

      Introduction

      Coenzyme Q (ubiquinone or Q)
      The abbreviations used are: Q
      coenzyme Q
      pABA
      para-aminobenzoic acid
      FMO
      flavin monooxygenase
      4-HB
      4-hydroxybenzoic acid
      3,4-diHB
      3,4-dihydroxybenzoic acid
      HHB
      3-hexaprenyl-4-hydroxybenzoic acid
      HAB
      3-hexaprenyl-4-aminobenzoic acid
      DHHB
      3-hexaprenyl-4,5-dihydroxybenzoic acid
      4-AP6
      3-hexaprenyl-4-aminophenol
      4-HP6
      3-hexaprenyl-4-hydroxyphenol
      HHAB
      3-hexaprenyl-4-amino-5-hydroxybenzoic acid
      IDMQ6
      4-imino-demethoxy-Q6
      DDMQ6
      demethyl-demethoxy-Q6
      DMQ6
      demethoxy-Q6
      pHBH
      para-hydroxybenzoate hydroxylase
      ECD
      electrochemical detection
      YNB
      yeast nitrogen base
      3H4AB
      3-hydroxy-4-aminobenzoic acid
      VA
      vanillic acid
      IDDMQ6
      4-imino-demethyl-demethoxy-Q6
      huCoq6
      human Coq6.
      is a redox-active lipid essential for electron and proton transport in the respiratory chain of mitochondria and some bacteria (
      • Bentinger M.
      • Tekle M.
      • Dallner G.
      Coenzyme Q-biosynthesis and functions.
      ,
      • Søballe B.
      • Poole R.K.
      Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management.
      ,
      • Aussel L.
      • Pierrel F.
      • Loiseau L.
      • Lombard M.
      • Fontecave M.
      • Barras F.
      Biosynthesis and physiology of coenzyme Q in bacteria.
      ). Q also serves as a membrane antioxidant and a cofactor of uncoupling proteins (
      • Bentinger M.
      • Tekle M.
      • Dallner G.
      Coenzyme Q-biosynthesis and functions.
      ) and has recently been implicated in osmoprotection in Escherichia coli (
      • Sévin D.C.
      • Sauer U.
      Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli.
      ). Q is composed of a fully substituted benzoquinone ring that is attached to a polyisoprenyl tail of various length (six isoprenyl units in Saccharomyces cerevisiae hence Q6, 10 units in humans, hence Q10). In S. cerevisiae, the biosynthesis of Q takes place in the mitochondrial matrix and implicates at least 12 proteins, Coq1-Coq9, Coq11, Arh1, and Yah1 (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ,
      • Allan C.M.
      • Awad A.M.
      • Johnson J.S.
      • Shirasaki D.I.
      • Wang C.
      • Blaby-Haas C.E.
      • Merchant S.S.
      • Loo J.A.
      • Clarke C.F.
      Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae.
      ), most of them being conserved in humans. Most Coq proteins form a multisubunit biosynthetic complex in S. cerevisiae (termed the CoQ-synthome), which is destabilized by the absence of a single Coq polypeptide, causing a drastic diminution of the steady state levels of some Coq proteins in Δcoq deletion mutants (
      • Hsieh E.J.
      • Gin P.
      • Gulmezian M.
      • Tran U.C.
      • Saiki R.
      • Marbois B.N.
      • Clarke C.F.
      Saccharomyces cerevisiae Coq9 polypeptide is a subunit of the mitochondrial coenzyme Q biosynthetic complex.
      ,
      • Tran U.C.
      • Clarke C.F.
      Endogenous synthesis of coenzyme Q in eukaryotes.
      ,
      • González-Mariscal I.
      • García-Testón E.
      • Padilla S.
      • Martín-Montalvo A.
      • Pomares-Viciana T.
      • Vazquez-Fonseca L.
      • Gandolfo-Domínguez P.
      • Santos-Ocaña C.
      Regulation of coenzyme Q biosynthesis in yeast: a new complex in the block.
      ). The instability of several Coq proteins in such mutants can be corrected through overexpression of the Coq8 kinase (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ) or by supplementing the growth medium with Q6 (
      • He C.H.
      • Xie L.X.
      • Allan C.M.
      • Tran U.C.
      • Clarke C.F.
      Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants.
      ). Under such stabilizing conditions, the Coq proteins are functional and allow accumulation of Q biosynthetic intermediates in Δcoq strains (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ), which provide information on the reaction deficient in a given Δcoq mutant.
      In 2010, Clarke's group and ourselves (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ,
      • Marbois B.
      • Xie L.X.
      • Choi S.
      • Hirano K.
      • Hyman K.
      • Clarke C.F.
      para-Aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae.
      ) discovered that S. cerevisiae is able to use para-aminobenzoic acid (pABA) in addition to 4-hydroxybenzoic acid (4-HB) as a precursor of Q6 (Fig. 1). Endogenous pABA and 4-HB originate from chorismate and are limiting for Q6 biosynthesis because their addition to the growth medium increases Q6 levels in S. cerevisiae (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ). pABA and 4-HB enter the Q6 biosynthetic pathway via the prenylation reaction catalyzed by Coq2 and then multiple enzymes modify the aromatic ring to yield Q6 (Fig. 1). Competition experiments demonstrated that pABA and 4-HB provided exogenously are equally efficient at promoting Q6 biosynthesis (
      • Marbois B.
      • Xie L.X.
      • Choi S.
      • Hirano K.
      • Hyman K.
      • Clarke C.F.
      para-Aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae.
      ). We also showed that several synthetic analogs of 4-HB (2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid (3,4-diHB), and vanillic acid (VA)) can serve as precursors of Q6 and can bypass deficient biosynthetic steps (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ,
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ). In particular, addition of VA or 3,4-diHB to the growth medium restores Q6 biosynthesis in Δcoq6 cells deficient for the C5-hydroxylation reaction (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ). Nevertheless, the effect of VA was annihilated by minute amounts of 4-HB. This result could either reflect an inefficient transport of VA to the mitochondrial matrix and/or a higher affinity of Coq2 for 4-HB than for VA (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ).
      Figure thumbnail gr1
      FIGURE 1S. 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.
      Coq6 catalyzes the C5-hydroxylation reaction (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ) and according to its primary sequence, it belongs to the family of class A flavoprotein monooxygenases (FMOs) (
      • van Berkel W.J.
      • Kamerbeek N.M.
      • Fraaije M.W.
      Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts.
      ). Class A FMOs contain a flavin adenine dinucleotide (FAD) and we recently demonstrated the presence of FAD in purified Coq6.
      A. Ismail, V. Leroux, M. Smaja, L. Gonzalez, M. Lombard, F. Pierrel, C. Mellot-Draznieks, and M. Fontecave, Coenzyme Q biosynthesis: evidence for a substrate access channel in the FAD-dependent monooxygenase Coq6. submitted for publication.
      The prototype enzyme of class A FMOs is para-hydroxybenzoate hydroxylase (
      • Huijbers M.M.
      • Montersino S.
      • Westphal A.H.
      • Tischler D.
      • van Berkel W.J.
      Flavin dependent monooxygenases.
      ), which catalyzes the ortho-hydroxylation of 4-HB in some bacteria (
      • Entsch B.
      • Cole L.J.
      • Ballou D.P.
      Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase.
      ). In para-hydroxybenzoate hydroxylase the FAD cofactor is reduced by NADPH and then reacts with dioxygen to form a FAD-hydroperoxide intermediate, which transfers a hydroxyl group onto the substrate (
      • Entsch B.
      • Cole L.J.
      • Ballou D.P.
      Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase.
      ,
      • Palfey B.A.
      • McDonald C.A.
      Control of catalysis in flavin-dependent monooxygenases.
      ). The resulting FAD-hydroxide then eliminates water, which results in oxidized FAD, the starting point of the next catalytic cycle. All class A FMOs proceed similarly and thus use one oxygen atom of the dioxygen molecule for hydroxylation of the substrate, whereas the other oxygen atom is reduced into water (
      • Huijbers M.M.
      • Montersino S.
      • Westphal A.H.
      • Tischler D.
      • van Berkel W.J.
      Flavin dependent monooxygenases.
      ). UbiI, UbiH, and UbiF are class A FMOs used by E. coli to catalyze the three hydroxylation reactions of Q8 biosynthesis (
      • Hajj Chehade M.
      • Loiseau L.
      • Lombard M.
      • Pecqueur L.
      • Ismail A.
      • Smadja M.
      • Golinelli-Pimpaneau B.
      • Mellot-Draznieks C.
      • Hamelin O.
      • Aussel L.
      • Kieffer-Jaquinod S.
      • Labessan N.
      • Barras F.
      • Fontecave M.
      • Pierrel F.
      ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation.
      ) and early in vivo labeling experiments indeed demonstrated the incorporation of molecular oxygen into three hydroxyl groups of Q8 (
      • Alexander K.
      • Young I.G.
      Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.
      ). In S. cerevisiae, only two hydroxylases are presently known (Fig. 1). The FMO Coq6 catalyzes the C5-hydroxylation (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ), whereas Coq7 catalyzes the C6-hydroxylation (
      • Stenmark P.
      • Grünler J.
      • Mattsson J.
      • Sindelar P.J.
      • Nordlund P.
      • Berthold D.A.
      A new member of the family of di-iron carboxylate proteins: Coq7 (clk-1), a membrane-bound hydroxylase involved in ubiquinone biosynthesis.
      ,
      • Tran U.C.
      • Marbois B.
      • Gin P.
      • Gulmezian M.
      • Jonassen T.
      • Clarke C.F.
      Complementation of Saccharomyces cerevisiae coq7 mutants by mitochondrial targeting of the Escherichia coli UbiF polypeptide: two functions of yeast Coq7 polypeptide in coenzyme Q biosynthesis.
      ) and uses a dinuclear iron center to activate dioxygen (
      • Behan R.K.
      • Lippard S.J.
      The aging-associated enzyme CLK-1 is a member of the carboxylate-bridged diiron family of proteins.
      ).
      To convert pABA into Q6, S. cerevisiae must replace the C4-amino group with a C4-hydroxyl group in a reaction termed C4-deamination (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ,
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ). Coq6 and Coq9 are thought to be important for the C4-deamination reaction because cells lacking either protein accumulate C4-amino containing intermediates when grown with exogenous pABA (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ,
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ,
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ). Δcoq6 cells overexpressing Coq8 (+ COQ8) form 3-hexaprenyl-4-aminophenol (4-AP6) in the presence of pABA and 3-hexaprenyl-4-hydroxyphenol (4-HP6) when 4-HB is used as a precursor (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ) (Fig. 1). The accumulation of 4-HP6 and 4-AP6 shows that the C1-decarboxylation and C1-hydroxylation reactions can take place in the absence of a C5-methoxy group. Δcoq9 + COQ8 cells produce 4-AP6 and 4-imino-demethoxy-Q6 (IDMQ6) with pABA, and 4-HP6 and demethoxy-Q6 (DMQ6) with 4-HB (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ) (Fig. 1). Thus deletion of Coq9 causes a partial inactivation of the C5-hydroxylation reaction catalyzed by Coq6 and a complete impairment of the C6-hydroxylation reaction catalyzed by Coq7 (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ). Accordingly, human Coq9 was demonstrated to bind lipids and to associate with Coq7, leading to the suggestion that Coq9 may present DMQ10 to Coq7 (
      • Lohman D.C.
      • Forouhar F.
      • Beebe E.T.
      • Stefely M.S.
      • Minogue C.E.
      • Ulbrich A.
      • Stefely J.A.
      • Sukumar S.
      • Luna-Sánchez M.
      • Jochem A.
      • Lew S.
      • Seetharaman J.
      • Xiao R.
      • Wang H.
      • Westphall M.S.
      • Wrobel R.L.
      • Everett J.K.
      • Mitchell J.C.
      • López L.C.
      • Coon J.J.
      • Tong L.
      • Pagliarini D.J.
      Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis.
      ). The role played by Coq6 and Coq9 in the yeast C4-deamination reaction is unclear and the step at which this reaction takes place is not defined (Fig. 1). The formation of IDMQ6 from pABA in Δcoq9 + COQ8 cells shows that all Coq biosynthetic enzymes up to Coq7 can accommodate substrates with a C4-amino group. However, the predominant accumulation of demethyl-demethoxy-Q6 (DDMQ6) and DMQ6 in Δcoq5 + COQ8 and Δcoq7 + COQ8 cells grown in the presence of pABA suggests that the C4-deamination reaction may take place prior to the C2-methyltransferase reaction catalyzed by Coq5 (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ).
      In this study, we address the question of the C4-deamination step allowing pABA conversion into Q6 in S. cerevisiae. We unambiguously show that this reaction is achieved by Coq6. We provide evidence supporting that the C4-deamination occurs on an early intermediate of Q6 biosynthetic pathway, namely 3-hexaprenyl-4-amino-5-hydroxybenzoic acid (HHAB) and that the reaction proceeds via hydroxylation of the C4-amino carbon atom of HHAB and subsequent loss of the amino group. We further show that Coq9 plays only an indirect role in the C4-deamination reaction, likely by affecting the C-terminal region of Coq6. Collectively, our results define Coq6 as an FMO that catalyzes two sequential hydroxylation reactions on two adjacent aromatic carbon atoms when pABA is used as a precursor of Q6 by S. cerevisiae.

      Experimental Procedures

       Plasmids and Chemicals

      Plasmids used in this study are listed in Table 1. pCOQ8 was constructed by amplifying the S. cerevisiae COQ8 gene from genomic DNA using the primers COQ8_3Xho (5′-GCTATTGGCAGAAGctcgagCGTTGCTAAG) and COQ8_5Eco (5′-GGTCTgaattcGATCCGGGTGTTCGG) for the PCR. The PCR product and the pRS423 plasmid were digested with EcoRI and XhoI, purified from agarose gel, and ligated. One plasmid containing the insert was checked by DNA sequencing. All chemicals were from Sigma. The synthesis of 13C7-pABA has been described (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ).
      TABLE 1Plasmids used in this study
      NameVector basePlasmid relevant genesCopy numberSource
      pCOQ8pRS423S. cerevisiae COQ8 under its own promoterHigh copyThis work
      pCOQ6 G386A-N388DpRS416S. cerevisiae COQ6 G386A-N388D under MET25 promoterLow copy
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      pCOQ6pCM189S. cerevisiae COQ6 under its own promoterLow copy
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      pCOQ6 M469XpCM189S. cerevisiae COQ6 M469X under its own promoterLow copy
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      pCOQ6 F420CpCM189S. cerevisiae COQ6 F420C under its own promoterLow copy
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      pCOQ6 A361DpCM189S. cerevisiae COQ6 A361D under its own promoterLow copy
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      pCOQ6 G248RpCM189S. cerevisiae COQ6 G248R under its own promoterLow copy
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      pCOQ6 L382EpCM189S. cerevisiae COQ6 L382E under its own promoterLow copy
      • Block A.
      • Widhalm J.R.
      • Fatihi A.
      • Cahoon R.E.
      • Wamboldt Y.
      • Elowsky C.
      • Mackenzie S.A.
      • Cahoon E.B.
      • Chapple C.
      • Dudareva N.
      • Basset G.J.
      The origin and biosynthesis of the benzenoid moiety of ubiquinone (coenzyme Q) in Arabidopsis.
      phuCOQ6pCM189Human COQ6 under CYC1 promoterLow copy
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.

       Yeast Strains and Culture Conditions

      S. cerevisiae strains used in this study are listed in Table 2. DNA transformations were performed with the PEG-lithium acetate method as previously reported (
      • Burke D.
      • Dawson D.
      • Stearns T.
      Methods in Yeast Genetics.
      ).
      TABLE 2S. cerevisiae strains used in this study
      StrainGenotypeSource
      W303MATα, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15R. Rothstein
      Δcoq6 (αW303 COQ6-2)MAT α, coq6::LEU2 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15
      • Gin P.
      • Hsu A.Y.
      • Rothman S.C.
      • Jonassen T.
      • Lee P.T.
      • Tzagoloff A.
      • Clarke C.F.
      The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis.
      W303 COQ6-2MAT a, coq6::LEU2 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15
      • Gin P.
      • Hsu A.Y.
      • Rothman S.C.
      • Jonassen T.
      • Lee P.T.
      • Tzagoloff A.
      • Clarke C.F.
      The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis.
      Δcoq6Δcoq9MAT a, coq6::LEU2 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 coq9::kanMXThis work
      Δcoq9MAT α, ylr201c::kanMX4 his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0Euroscarf
      The cells were grown in yeast nitrogen base (YNB) without pABA and folate (YNB-p) from MP Biomedicals. YNB-p was supplemented with 10 μm FeCl3, with carbon sources (glucose, galactose, or lactate/glycerol at 2% (w/v)) and with amino acids and nucleobases to cover the yeast auxotrophies except for the selectin of plasmids. For solid media, Bacto-agar (Euromedex) was added at 1.6% (w/v). Typically, strains were incubated overnight at 30 °C, 180 rpm in 2 ml of medium containing 2% (w/v) galactose and 0.2% (w/v) glucose. This preculture was diluted 100-fold in 5 ml of medium containing 2% (w/v) galactose and the cells were cultured at 30 °C at 180 rpm until A600 reached ∼2. The cells were either used for serial dilution growth assay or for quinone content analysis. For mitochondrial preparations, yeast cells were grown at 30 °C in rich YPGal medium containing 2% (w/v) bactopeptone (Difco), 1% (w/v) yeast extract (Difco), and 2% (w/v) galactose.
      The Δcoq6Δcoq9 strain was constructed by transforming the W303 COQ6-2 strain with a PCR product corresponding to the kanMX4 cassette flanked by the COQ9 promoter and terminator regions. This PCR product was obtained with primers COQ9_5 (5′-GGCGGAAGAAAAATAGC) and COQ9_3 (5′-CCACTGGCCCAGGAAGG) used on the genomic DNA from the Δcoq9 strain. Transformants were selected on YPD-agar plates containing G418 and clones were checked by coq9 locus PCR and phenotypic characterization.

       Analysis of the Quinone Content

      The cultures were placed on ice for 30 min, then the cells were collected by centrifugation, washed once with ice-cold water, and their wet weight was determined in pre-weighted Eppendorf tubes before freezing at −20 °C. For lipid extraction, glass beads (100 μl), 50 μl of KCl (0.15 m), a Q4 solution (4 μm in methanol, 2 μl/mg of wet weight), and 0.6 ml of methanol were added to cell pellets (10–30 mg wet weight) and the tubes were vortexed for 10 min. Neutral lipids were extracted by adding 0.4 ml of petroleum ether (40–60 °C boiling range) and by vortexing for 3 min. The phases were separated by centrifugation (3 min, 1000 × g at room temperature). The upper petroleum ether layer was transferred to a fresh tube. Petroleum ether (0.4 ml) was added to the glass beads and methanol-containing tube, and the extraction was repeated once more. The petroleum ether layers were combined and dried under argon. The lipids were resuspended in 100 μl of methanol, and aliquots were analyzed by reversed-phase high-pressure liquid chromatography (HPLC) on a Dionex U3000 system equipped with a C18 column (Betabasic-18, 5 μm, 4.6 × 150 mm, Thermo Scientific) at a flow rate of 1 ml/min with a mobile phase composed of 75% (98% (v/v) methanol, 2% (v/v) 1 m ammonium acetate), 5% isopropyl alcohol, and 20% acetonitrile. Hydroquinones present in injected samples were oxidized with a precolumn 5020 guard cell set in oxidizing mode (E, +600 mV). Electrochemical detection (ECD) was performed with a Coulochem III (ESA) equipped with a 5011A analytical cell (E1, −550 mV; E2, 550 mV). The standard Q4 solution was injected in the same conditions to generate a standard curve that was used to correct for sample loss during the organic extraction (on the basis of the recovery of the Q4 internal standard) and to quantify Q6. When mass spectrometry (MS) detection was needed, the flow was split after the diode array detector with an adjustable split valve (Analytical Scientific Instruments) to allow simultaneous EC (60% of the flow) and MS (40% of the flow) detections. MS detection was achieved with an MSQ Plus spectrometer (Thermo) used in positive mode with electrospray ionization. The probe temperature was 450 °C and the cone voltage was 80 V. Because of the precolumn guard cell, the following compounds were detected by single ion monitoring in their oxidized state: 4-AP6 (M + H+), m/z 515.9–516.9, 7–9 min, scan time 0.2 s; 13C6-4-AP6 (M + H+), m/z 521.9–522.9, 7–9 min, scan time 0.2 s; IDMQ6 (M + H+), m/z 559.9–560.9, 9.5–14 min, scan time 0.2 s; 13C6-IDMQ6 (M + H+), m/z 565.9–566.9, 9.5–14 min, scan time 0.2 s; DMQ6 (M + NH4+), m/z 577.9–578.9, 7–12 min, scan time 0.6 s; 13C6-DMQ6 (M + NH4+), m/z 583.9–584.9, 7–12 min, scan time 0.6 s; Q6 (M + NH4+), m/z 607.9–608.9, 9–11.5 min, scan time 0.2 s. MS spectra were also recorded between m/z 500 and 700 or 450 and 650 with a scan time of 0.4 s.

       Mitochondrial Preparation and Western Blotting

      Yeast cells grown on YPGal were harvested in the late log phase (A600 near 5). Mitochondria were prepared as described by Daum et al. (
      • Daum G.
      • Böhni P.C.
      • Schatz G.
      Import of proteins into mitochondria: cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria.
      ). Briefly, spheroplasts obtained after enzymatic digestion of the cell wall by Zymolyase 20T (Seikagaku) were disrupted by Dounce homogenization in 0.6 m mannitol, 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 0.1% (w/v) BSA, and 1 mm PMSF. Mitochondria were isolated by differential centrifugation, washed in the same buffer devoid of BSA and PMSF, and stored in liquid nitrogen. The protein concentrations were determined using a BCA protein assay kit (Sigma) and bovine serum albumin as a standard. 20 μg of mitochondrial proteins were heated at 95 °C for 5 min in Laemmli buffer and separated on a 12.5% acrylamide SDS-PAGE gel. Antibodies were used at the following dilutions: anti-C-terminal peptide of Anc2p (
      • Clémençon B.
      • Rey M.
      • Dianoux A.C.
      • Trézéguet V.
      • Lauquin G.J.
      • Brandolin G.
      • Pelosi L.
      Structure-function relationships of the C-terminal end of the Saccharomyces cerevisiae ADP/ATP carrier isoform 2.
      ), 1/3000; anti-SDS-treated Por1 (
      • Clémençon B.
      • Rey M.
      • Dianoux A.C.
      • Trézéguet V.
      • Lauquin G.J.
      • Brandolin G.
      • Pelosi L.
      Structure-function relationships of the C-terminal end of the Saccharomyces cerevisiae ADP/ATP carrier isoform 2.
      ), 1/2000; and anti-Coq6 (
      • Gin P.
      • Hsu A.Y.
      • Rothman S.C.
      • Jonassen T.
      • Lee P.T.
      • Tzagoloff A.
      • Clarke C.F.
      The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis.
      ), 1/500. Immunodetection was performed using horseradish peroxidase-coupled protein A and the ECL-enhanced chemiluminescence system (Amersham Biosciences).

       18O2 Labeling Experiment

      In a 10-ml culture flask, 1 ml of lactate/glycerol YNB-p medium supplemented with 20 μm 4-HB or 20 μm pABA was inoculated with 10 μl of an overnight preculture of W303 in the same medium without precursors. The culture flask was extensively degassed with N2 and sealed with a septum prior to the introduction of 2.5 ml of 18O2 (97% enrichment, eurisotop) with a syringe. The culture was incubated at 30 °C, 200 rpm for 24 h until the cells reached saturation. The cells were then placed on ice for 30 min before opening the flask to harvest the cells.

       Model of Coq6

      The homology model of wild-type Coq6 was taken from our recent experimental and computational study of the Coq6-FAD complex.6 This model was constructed using MODELLER (
      • Sali A.
      • Blundell T.L.
      Comparative protein modelling by satisfaction of spatial restraints.
      ) on the basis of three functionally related flavoprotein monooxygenase PDB structures: 2X3N (pqsL, an alkylquinolone hydroxylase from Pseudomonas aerguinosa), 4N9X (a ubiquinone biosynthesis hydroxylase from Erwinia carotovora), and 4K22 (ubiI, a ubiquinone biosynthesis hydroxylase from E. coli). Structural models of mutated Coq6 enzyme were generated on the basis of this wild-type model. All models were subjected to molecular dynamics using the AMBER99SB-ILDN force field (
      • Lindorff-Larsen K.
      • Piana S.
      • Palmo K.
      • Maragakis P.
      • Klepeis J.L.
      • Dror R.O.
      • Shaw D.E.
      Improved side-chain torsion potentials for the Amber ff99SB protein force field.
      ) as implemented in GROMACS 4.6.5 (
      • Pronk S.
      • Páll S.
      • Schulz R.
      • Larsson P.
      • Bjelkmar P.
      • Apostolov R.
      • Shirts M.R.
      • Smith J.C.
      • Kasson P.M.
      • van der Spoel D.
      • Hess B.
      • Lindahl E.
      GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit.
      ). 20-ns production trajectories were analyzed with CAVER (
      • Chovancova E.
      • Pavelka A.
      • Benes P.
      • Strnad O.
      • Brezovsky J.
      • Kozlikova B.
      • Gora A.
      • Sustr V.
      • Klvana M.
      • Medek P.
      • Biedermannova L.
      • Sochor J.
      • Damborsky J.
      CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures.
      ) and VMD (
      • Humphrey W.
      • Dalke A.
      • Schulten K.
      VMD: visual molecular dynamics.
      ) to detect channels leading from the protein surface to the active site.

      Results

       The Hydroxyl Group on C4 of Q6 Originates from Dioxygen in pABA-grown Cells

      It was previously hypothesized that the C4-deamination reaction may occur via Schiff base chemistry with a water molecule attacking the C4-imino group of an oxidized Q6 biosynthetic intermediate (
      • Marbois B.
      • Xie L.X.
      • Choi S.
      • Hirano K.
      • Hyman K.
      • Clarke C.F.
      para-Aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae.
      ). To test this hypothesis, we cultivated wild-type S. cerevisiae W303 cells in medium containing 75% H218O (v/v) and supplemented with either 50 μm 4-HB or pABA. In both cases, no labeling was detected in Q6 (C39H58O4) because the main ions of the mass spectrum were at m/z 608 (M + NH4+) and m/z 591 (M + H+) (Fig. 2A and data not shown). This result establishes that water is not the source of the C4-hydroxyl group of Q6 in pABA-grown cells. We then cultured the W303 strain in medium supplemented with 50 μm 4-HB or pABA in an atmosphere containing ∼80% N2 (v/v) and 20% 18O2 (v/v). From the 4-HB culture, the mass spectrum of Q6 showed two main ions at m/z 614.3 (M + NH4+) and 597.3 (M + H+) (Fig. 2B), which correspond to an increase of 6 units of the mass of Q6 (C39H5816O18O3) compared with unlabeled Q6 (C39H5816O4) (Fig. 2A). This result demonstrates for the first time that the three hydroxylation reactions of the S. cerevisiae Q6 biosynthetic pathway use dioxygen as a substrate in agreement with results previously obtained in E. coli (
      • Alexander K.
      • Young I.G.
      Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.
      ). In the case of the pABA culture, the mass spectrum of Q6 showed prominent ions at m/z 616.3 (M + NH4+) and 599.3 (M + NH4+) corresponding to a Q6 molecule in which all four oxygen atoms are labeled (C39H5818O4)(Fig. 2C). These results unambiguously show that an additional 18O atom is incorporated into Q6 when pABA is used as a precursor as compared with 4-HB and establish that dioxygen is the source of the OH group on C4, which substitutes the amino group originally present on pABA. This reaction thus relies on a protein that has the capacity to activate dioxygen.
      Figure thumbnail gr2
      FIGURE 2Isotopic 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).

       Metabolism of 3-Hydroxy-4-aminobenzoic Acid into Q6 Requires an Active Coq6

      To verify whether the C4-deamination defect is a consequence or not of the C5-hydroxylation defect in coq6-deficient cells, 3-hydroxy-4-aminobenzoic acid (3H4AB) was used as substrate. Indeed, if prenylated by Coq2, 3H4AB will yield HHAB with a C5-hydroxyl group without assistance by Coq6. First, we established that exogenous 3H4AB is indeed prenylated by Coq2 because its addition at 1 mm in the growth medium was able to increase the cellular Q6 content of W303 cells grown in pABA-free medium to levels similar to those obtained with 10 μm 4-HB (Fig. 3, A and B). Therefore, 3H4AB is a precursor of Q6 in WT cells. Besides Q6, only a minute amount of DMQ6 was detected by HPLC-ECD in extracts from 3H4AB-treated cells (Fig. 3A), showing that the C4-amino group does not perturb a particular biosynthetic step. In fact, C4-deamination of compounds derived from 3H4AB also implicated O2 because cells grown with 3H4AB in 80% N2 and 20% 18O2 synthesized mostly 18O3-Q6 (Fig. 3C), consistent with 18O labeling at positions C1, C4, and C6 and with the absence of labeling on the C5-hydroxyl derived from 3H4AB. In Δcoq6 + pCOQ8 cells, 4-HB caused the accumulation of 4-HP6, whereas 3,4-diHB restored Q6 biosynthesis, as already shown (Fig. 3D) (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ). In contrast, 3H4AB was unable to yield any Q6 but several compounds were detected instead: 4-HP6, DMQ6, and IDMQ6 (Fig. 3D). 4-HP6 likely originates from endogenous 4-HB, showing that exogenous 3H4AB cannot completely outcompete endogenous 4-HB, as already established for vanillic acid (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ). The most prominent compound that eluted at 11.6 min (Fig. 3D) was identified as IDMQ6 based on its mass spectrum (M + H+) m/z 560.2 (Fig. 3E) and on its co-elution with the previously characterized IDMQ6 molecule from Δcoq9 +COQ8 cells grown in the presence of pABA (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ). The accumulation of IDMQ6 demonstrates that the C4-deamination is impaired in Δcoq6 + pCOQ8 cells but not completely because DMQ6 was also detected (Fig. 3D). DMQ6 and IDMQ6 are the most downstream product of the Q6 pathway detected in Δcoq6 + pCOQ8 cells grown in the presence of 3H4AB, showing that the C6-hydroxylation by Coq7 is inhibited in these conditions. IDMQ6 may be responsible for this inhibition in agreement with the proposition that an intermediate derived from prenyl-pABA has an inhibitory effect on the C6-hydroxylation of DMQ6 (
      • Marbois B.
      • Xie L.X.
      • Choi S.
      • Hirano K.
      • Hyman K.
      • Clarke C.F.
      para-Aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae.
      ). To verify that the results obtained in Δcoq6 cells were independent from the overexpression of Coq8, we next tested the effect of 3H4AB in cells expressing Coq6 G386A-N388D. These point mutations inactivate Coq6 but do not destabilize the protein that allows for assembly of the CoQ-synthome without Coq8 overexpression (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ). Like in Δcoq6 + pCOQ8 cells, 3,4-diHB resulted in Q6 biosynthesis in Δcoq6 + pCOQ6 G386A/N388D but 3H4AB failed and caused mostly the accumulation of IDMQ6 (Fig. 3F). The electroactive compound co-eluting with Q4 at 4.3 min in 3H4AB-treated cells is likely to be oxidized prenylated 3H4AB based on its mass spectrum (M + H+) m/z 560.1 (data not shown) and on its short retention time on the reverse phase column caused by the polarity of the carboxyl group. Overall, the accumulation of IDMQ6 in coq6-deficient cells grown in the presence of 3H4AB shows that (i) the conversion of this substrate analog into Q6 is dependent of an active Coq6 enzyme, (ii) the presence of a methoxy group on C5 is not sufficient to promote efficient C4-deamination in the absence of an active Coq6 enzyme. These results establish that the lack of C4-deamination in coq6-deficient cells grown in pABA (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ) is not a mere consequence of the absence of the methoxy group on C5 but rather support that Coq6 itself is required for efficient C4-deamination.
      Figure thumbnail gr3
      FIGURE 3Conversion 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.

       L382E and M469X Mutations in Coq6 Affect the C4-deamination While Preserving Partially the C5-hydroxylation

      We next tested the efficiency of five Coq6 point mutants to synthesize Q6 from 4-HB or pABA. In this last case, pABA added in excess in the medium competes efficiently with endogenous 4-HB to enter the Q biosynthetic pathway (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ). The mutations G248R, A361D, F420C, and M469X (truncation of the last 11 residues) in S. cerevisiae Coq6 correspond to pathological mutations found in the human Coq6 protein (huCoq6) (
      • Heeringa S.F.
      • Chernin G.
      • Chaki M.
      • Zhou W.
      • Sloan A.J.
      • Ji Z.
      • Xie L.X.
      • Salviati L.
      • Hurd T.W.
      • Vega-Warner V.
      • Killen P.D.
      • Raphael Y.
      • Ashraf S.
      • Ovunc B.
      • Schoeb D.S.
      • McLaughlin H.M.
      • Airik R.
      • Vlangos C.N.
      • Gbadegesin R.
      • Hinkes B.
      • Saisawat P.
      • Trevisson E.
      • Doimo M.
      • Casarin A.
      • Pertegato V.
      • Giorgi G.
      • Prokisch H.
      • Rötig A.
      • Nürnberg G.
      • Becker C.
      • Wang S.
      • Ozaltin F.
      • Topaloglu R.
      • Bakkaloglu A.
      • Bakkaloglu S.A.
      • Müller D.
      • Beissert A.
      • Mir S.
      • Berdeli A.
      • Varpizen S.
      • Zenker M.
      • Matejas V.
      • Santos-Ocaña C.
      • Navas P.
      • Kusakabe T.
      • Kispert A.
      • Akman S.
      • Soliman N.A.
      • Krick S.
      • Mundel P.
      • Reiser J.
      • Nürnberg P.
      • Clarke C.F.
      • Wiggins R.C.
      • Faul C.
      • Hildebrandt F.
      COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness.
      ,
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      ) and decrease the Q6 content to different extents in cells grown in rich medium (
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      ). We recently constructed a L382E mutant that retains significant C5-hydroxylase activity with 4-HB added to the growth medium.6 In our molecular model of Coq6,6 the Ala-361 and Phe-420 residues are far from the FAD molecule that is buried inside the protein and from the predicted active site that faces the isoalloxazine ring of FAD (Fig. 4A). Conversely, the Leu-382 and Gly-248 residues face each other across the substrate access channel (Fig. 4B), which leads to the active site and likely accommodates the hexaprenyl tail of Coq6 substrates.6 The last 11 residues of Coq6, which are truncated in the M469X mutant, are localized on the protein surface (volume highlighted in red, Fig. 4A) and are also in close proximity to the substrate access channel (Fig. 4B). The growth of S. cerevisiae on a respiratory medium is dependent on the function of the mitochondrial respiratory chain and thus on the biosynthesis of Q6. Δcoq6 strains containing pCOQ6, pCOQ6 A361D, or pCOQ6 F420C grew equally well on the respiratory medium lactate/glycerol containing either 4-HB or pABA (Fig. 4C). In contrast, the growth of strains with the plasmids pCOQ6 M469X, pCOQ6 G248R, or pCOQ6 L382E was superior when the medium was supplemented with 4-HB as compared with pABA (Fig. 4C). The growth pattern of all strains was similar whether media were supplemented or not with 4-HB (Fig. 4C), showing that Q6 synthesized from endogenous substrates is not limiting for respiratory growth and supporting that the endogenous substrate predominantly used for Q6 biosynthesis is 4-HB. This last point is in line with the major accumulation of 4-HP6 over 4-AP6 in Δcoq6 + pCOQ8 cells grown in pABA-free medium (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ). HPLC-ECD analysis showed that pABA or 4-HB yielded a comparable Q6 content in Δcoq6 cells expressing Coq6, Coq6 A361D, or Coq6 F420C (Fig. 4D). In contrast, Coq6 M469X led to the synthesis of an appreciable amount of Q6 when 4-HB was added to the growth medium but Q6 was almost undetectable with pABA as a precursor (Fig. 4E). Instead, 4-AP6, IDMQ6, and a small quantity of DMQ6 were detected (Fig. 4E). The accumulation of 4-AP6 shows that the C5-hydroxylation of HAB is perturbed and the predominant synthesis of C4-amino intermediates over C4-hydroxy intermediates demonstrates that the C4-deamination is almost completely abrogated by the M469X truncation. Similarly, the L382E mutant was efficient at synthesizing Q6 from 4-HB but the main compound accumulated from pABA was IDMQ6 (Fig. 4E), demonstrating that the C5-hydroxylation was functional and C4-deamination was limiting in this strain. The G248R mutation had more drastic effects because the amount of Q6 synthesized from 4-HB was minimal, whereas from pABA, 4-AP6 and a minute amount of IDMQ6 were detected (Fig. 4E). Based on the synthesis of Q6 and IDMQ6 in the presence of 4-HB and pABA, respectively, we conclude that Coq6 M469X and Coq6 L382E are competent for C5-hydroxylation of HHB and HAB. In contrast, the C4-deamination is limiting because IDMQ6 is accumulated predominantly over DMQ6 and Q6 in cells grown in the presence of pABA (Fig. 4E). Altogether, these results show that the M469X truncation and the L382E mutation impact slightly the C5-hydroxylation but strongly the C4-deamination.
      Figure thumbnail gr4
      FIGURE 4Point 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.

       Coq9 Is Indispensable to the Activity of Coq6 M469X

      We reported a defect in C4-deamination not only in cells lacking Coq6, but also in cells lacking Coq9 (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ). However, the absence of Coq9 affects Coq6 and Coq7 activities leading to accumulation of 4-HP6 and DMQ6 in the presence of 4-HB and to 4-AP6 and IDMQ6 in the presence of pABA (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ). Therefore, the defect in C4-deamination in Δcoq9 cells may indirectly result from the perturbation of Coq6. As anticipated, expression of Coq6 in Δcoq6Δcoq9 cells grown in the presence of 4-HB caused the biosynthesis of 4-HP6 and DMQ6 (Fig. 5A), showing that the C5-hydroxylation catalyzed by Coq6 was partly impaired. Unexpectedly, DMQ6 was undetectable in Δcoq6Δcoq9 cells expressing Coq6 M469X (Fig. 5A), demonstrating that the C5-hydroxylation was completely abrogated. As judged from immunodetection on purified mitochondria, Coq9 is not required for the stability of Coq6 M469X because the steady state levels of Coq6 and Coq6 M469X were comparable whether Coq9 was present or not (Fig. 5B). Because Coq6 M469X is functional in cells containing Coq9 grown with 4-HB (Fig. 4E), we conclude that the absence of Coq9 and the truncation of Coq6 have an additive negative impact on the C5-hydroxylation. Likewise, in the presence of pABA, IDMQ6 was synthesized by Δcoq6Δcoq9 cells expressing Coq6 but not Coq6 M469X (Fig. 5A). Together, our results show that the presence of Coq9 is indispensable to the C5-hydroxylation activity of Coq6 lacking its C-terminal part, which we also showed to be important for the C4-deamination reaction (Fig. 4E). Thus, the C4-deamination defect observed in Δcoq9 cells (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ) likely results from the perturbation of Coq6.
      Figure thumbnail gr5
      FIGURE 5Genetic 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.

       Human Coq6 Supports C4-deamination in Yeast Cells Lacking Coq9

      Several reports document that pABA is not a precursor of Q10 in different human cell lines (
      • Duberley K.E.
      • Abramov A.Y.
      • Chalasani A.
      • Heales S.J.
      • Rahman S.
      • Hargreaves I.P.
      Human neuronal coenzyme Q10 deficiency results in global loss of mitochondrial respiratory chain activity, increased mitochondrial oxidative stress and reversal of ATP synthase activity: implications for pathogenesis and treatment.
      ,
      • González-Aragón D.
      • Burón M.I.
      • López-Lluch G.
      • Hermán M.D.
      • Gómez-Díaz C.
      • Navas P.
      • Villalba J.M.
      Coenzyme Q and the regulation of intracellular steady-state levels of superoxide in HL-60 cells.
      ,
      • Xie L.X.
      • Williams K.J.
      • He C.H.
      • Weng E.
      • Khong S.
      • Rose T.E.
      • Kwon O.
      • Bensinger S.J.
      • Marbois B.N.
      • Clarke C.F.
      Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis.
      ). Because huCoq6 expressed in S. cerevisiae Δcoq6 cells partially complements the C5-hydroxylation defect (
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      ), we tested the effect of the addition of pABA to the growth medium. The complementation of the respiratory growth defect of Δcoq6 cells by huCoq6 was efficient when the medium contained 4-HB but not pABA (Fig. 6A). HPLC-ECD analysis revealed that the Q6 content was indeed very low in Δcoq6 + phuCOQ6 cells grown in the presence of pABA compared with 4-HB (Fig. 6B). 4-AP6 accumulated predominantly but IDMQ6 was also detected (Fig. 6B), indicating that the C5-hydroxylation took place without C4-deamination. Although huCoq6 is much less efficient than yeast Coq6 at restoring Q6 biosynthesis in Δcoq6 cells (Ref.
      • Doimo M.
      • Trevisson E.
      • Airik R.
      • Bergdoll M.
      • Santos-Ocaña C.
      • Hildebrandt F.
      • Navas P.
      • Pierrel F.
      • Salviati L.
      Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q deficiency.
      and compare Figs. 4D and 6B), phuCoq6 yielded a higher DMQ6 content than pCoq6 in Δcoq6Δcoq9 cells grown in the presence of 4-HB (compare Figs. 5A and 6C). In the presence of pABA, IDMQ6 was increased in Δcoq6Δcoq9 + phuCoq6 cells compared with Δcoq6 + phuCoq6 cells (compare Fig. 6, B and C), establishing that the presence of Coq9 is in fact detrimental to the C5-hydroxylase activity of huCoq6. In addition to IDMQ6 and besides 4-AP6, a large peak of DMQ6 was apparent in pABA-treated Δcoq6Δcoq9 + phuCoq6 cells (Fig. 6C). To verify the origin of DMQ6, we grew these cells in the presence of pABA or 13C7-pABA and analyzed the quinone content and the labeling in each compound by HPLC-ECD-MS. The quantities of 4-AP6, DMQ6, and IDMQ6 formed in pABA- or 13C7-pABA-treated cells were comparable and, as expected, 4-AP6 and IDMQ6 were almost entirely labeled (99%) in the 13C7-pABA culture (Fig. 6D). In these conditions, 13C6-DMQ6 represented 71% of the total DMQ6 (Fig. 6D), proving that the majority of DMQ6 originated from the exogenously added 13C7-pABA. Altogether, these results establish that Coq9 prevents huCoq6 from functioning efficiently in S. cerevisiae and show that, in the absence of Coq9, huCoq6 is able to support both the C5-hydroxylation and the C4-deamination.
      Figure thumbnail gr6
      FIGURE 6Human 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.

      Discussion

       Coq6 Is Responsible for the C4-deamination

      In 2010, it was discovered that S. cerevisiae is able to use pABA in addition to 4-HB as a precursor of Q6 (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ,
      • Marbois B.
      • Xie L.X.
      • Choi S.
      • Hirano K.
      • Hyman K.
      • Clarke C.F.
      para-Aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae.
      ). The conversion of pABA into Q6 requires a C4-deamination reaction in which the C4-amino group is replaced by a C4-hydroxyl group. The results presented in this study strongly support that Coq6, the monooxygenase responsible for the C5-hydroxylation, fulfills also the C4-deamination. This conclusion relies on the following observations: (i) coq6 mutant cells are defective not only in C5-hydroxylation but also in C4-deamination whether pABA or its C5-hydroxylated analog, 3H4AB, are provided as a substrate in the growth medium (Fig. 3). (ii) Mutation of Leu-382, a residue buried inside Coq6, severely affects the C4-deamination reaction, whereas the C5-hydroxylation is mostly maintained (Fig. 4). (iii) huCoq6 is able to complement the C5-hydroxylation and the C4-deamination defects of S. cerevisiae Δcoq6 cells, albeit only in the absence of the Coq9 protein (Fig. 6). The function of Coq9 with respect to Coq6 activity is discussed below. (iv) The oxygen atom introduced at C4 exclusively derives from molecular oxygen (Fig. 2), in agreement with the involvement of an O2 activating enzyme in the deamination reaction.

       Proposed Mechanism of the Dioxygen-mediated Deamination

      Our 18O2 in vivo labeling experiments confirm that dioxygen is the substrate of the three hydroxylation reactions (on C1, C5, and C6) necessary to synthesize Q6 from 4-HB, which implies that the unknown C1-hydroxylase is likely also a dioxygen-activating enzyme. The 18O2 labeling experiment with pABA as a substrate unambiguously shows that Q6 incorporates four 18O atoms, demonstrating that dioxygen is also the source of the oxygen atom at C4, which is introduced during the C4-deamination reaction. We thus propose a unifying mechanism for the C5-hydroxylation and the C4-deamination by Coq6 in agreement with the classical mechanism of class A FMOs to which Coq6 belongs (
      • Palfey B.A.
      • McDonald C.A.
      Control of catalysis in flavin-dependent monooxygenases.
      ). FAD is first reduced by NAD(P)H and reacts with dioxygen to form a flavin C4a-hydroperoxide adduct. The hydroxylation reaction proceeds via a nucleophilic attack of the C5 carbon atom of HAB onto the distal oxygen of the peroxide (Scheme 1, step A). Then, aromatization of the product occurs by deprotonation leading to the final phenol product (HHAB), whereas the oxidized flavin is regenerated by elimination of a water molecule from the flavin C4a-hydroxide (Scheme 1, step B). This reaction mechanism shown for the C4-amino substrate (HAB) (Scheme 1) also applies to the C4-hydroxy substrate (HHB) produced when 4-HB and not pABA is prenylated by Coq2 (Fig. 1). Next, the C4-deamination proceeds also via a nucleophilic attack of the C4 carbon atom of the HHAB intermediate onto the distal oxygen of the FAD-hydroperoxide (Scheme 1, step A′). The geminal amino and hydroxy groups at C4 easily convert to a keto function by elimination of NH3 facilitated by protonation of the amino group, thus generating an ortho-quinone intermediate (Scheme 1, steps B′ and C′). The latter is easily reducible into DHHB. Although the reducing system for this reaction is unknown, we raise the possibility that the reduced FAD cofactor of Coq6 is involved via hydride transfer to the quinone. Reduced flavins have indeed been extensively studied as quinone reducing agents (
      • Swanson M.A.
      • Usselman R.J.
      • Frerman F.E.
      • Eaton G.R.
      • Eaton S.S.
      The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein.
      ,
      • Singh H.
      • Arentson B.W.
      • Becker D.F.
      • Tanner J.J.
      Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site.
      ,
      • Anusevicius Z.
      • Miseviciene L.
      • Medina M.
      • Martinez-Julvez M.
      • Gomez-Moreno C.
      • Cenas N.
      FAD semiquinone stability regulates single- and two-electron reduction of quinones by Anabaena PCC7119 ferredoxin: NADP+ reductase and its Glu301Ala mutant.
      ). Alternatively, the ferredoxin/ferredoxin reductase system (Yah1/Arh1) (
      • Pierrel F.
      • Hamelin O.
      • Douki T.
      • Kieffer-Jaquinod S.
      • Mühlenhoff U.
      • Ozeir M.
      • Lill R.
      • Fontecave M.
      Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
      ) may be implicated in the reduction of DHHB. Overall, our results seem to indicate that Coq6 is a unique multifunctional redox enzyme responsible for O2-dependent oxygen atom insertion at two different positions of an aromatic ring as well as possibly for reduction of a quinone. The proposed mechanism also accounts for the labeling obtained when 3H4AB was provided as substrate (Fig. 3C) because prenylation of this compound yields HHAB in which the C5-hydroxyl will not be labeled in vivo when derived from 3H4AB. Our proposed mechanism ought to be confirmed by in vitro experiments. Despite extensive efforts, we have not so far obtained in vitro activity for purified Coq6, possibly because interacting partners of the in vivo CoQ-synthome (
      • Allan C.M.
      • Awad A.M.
      • Johnson J.S.
      • Shirasaki D.I.
      • Wang C.
      • Blaby-Haas C.E.
      • Merchant S.S.
      • Loo J.A.
      • Clarke C.F.
      Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae.
      ,
      • Hsieh E.J.
      • Gin P.
      • Gulmezian M.
      • Tran U.C.
      • Saiki R.
      • Marbois B.N.
      • Clarke C.F.
      Saccharomyces cerevisiae Coq9 polypeptide is a subunit of the mitochondrial coenzyme Q biosynthetic complex.
      ) may be required for Coq6 to adopt its functional tridimensional structure, contrary the other class A FMOs studied to date.
      Figure thumbnail grs1
      SCHEME 1Proposed 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.

       Different Fates for the Amino Group on Aromatic Substrates of FMOs

      Anthranilate hydroxylase is an FMO that hydroxylates anthranilate at position C3 and at the same time deaminates position C2 in a single reaction, resulting in the introduction of two oxygen atoms and thus formation of 2,3-dihydroxybenzoic acid (
      • Powlowski J.B.
      • Dagley S.
      • Massey V.
      • Ballou D.P.
      Properties of anthranilate hydroxylase (deaminating), a flavoprotein from Trichosporon cutaneum.
      ). Labeling experiments established that the C3 hydroxyl derives from dioxygen, whereas the C2 hydroxyl derives from solvent water (
      • Powlowski J.B.
      • Dagley S.
      • Massey V.
      • Ballou D.P.
      Properties of anthranilate hydroxylase (deaminating), a flavoprotein from Trichosporon cutaneum.
      ). The deamination reaction catalyzed by anthranilate hydroxylase was therefore proposed to occur via nucleophilic substitution of the amino group by a water molecule during the course of the oxidation (
      • Powlowski J.B.
      • Dagley S.
      • Massey V.
      • Ballou D.P.
      Properties of anthranilate hydroxylase (deaminating), a flavoprotein from Trichosporon cutaneum.
      ). Such a mechanism is clearly not possible in the case of Coq6 considering the results of our experiments with 18O2 and H218O. Another FMO, kynurenine 3-monooxygenase, catalyzes an aromatic hydroxylation ortho to an amino group without hydrolyzing it (
      • Crozier-Reabe K.R.
      • Phillips R.S.
      • Moran G.R.
      Kynurenine 3-monooxygenase from Pseudomonas fluorescens: substrate-like inhibitors both stimulate flavin reduction and stabilize the flavin-peroxo intermediate yet result in the production of hydrogen peroxide.
      ). Thus, the amino group ortho to the position hydroxylated by different FMOs encounters different fates: it can either remain unaffected, be hydrolyzed by a water molecule, or be replaced by a hydroxyl group derived from dioxygen as in the case of Coq6. Because all these FMOs utilize the same chemistry for hydroxylation, i.e. the nucleophilic attack of the substrate onto the electrophilic FAD-hydroperoxo intermediate, the different outcomes regarding the amino group likely results from subtle differences between the proteins, like solvent accessibility to the active site and positioning of the amino group with respect to the FAD-hydroperoxo.

       Regioselectivity of FMOs

      Most monooxygenases are highly regiospecific, however, a few enzymes are known to catalyze sequential hydroxylations at separated sites (
      • Cochrane R.V.
      • Vederas J.C.
      Highly selective but multifunctional oxygenases in secondary metabolism.
      ). For example, the FMO PgaE from Streptomyces is responsible for two consecutive hydroxylation reactions in the formation of gaudamycin C (
      • Kallio P.
      • Patrikainen P.
      • Suomela J.-P.
      • Mäntsälä P.
      • Metsä-Ketelä M.
      • Niemi J.
      Flavoprotein hydroxylase PgaE catalyzes two consecutive oxygen-dependent tailoring reactions in angucycline biosynthesis.
      ). The large active site cavity revealed by the crystal structure of PgaE is compatible with the two alternative substrates binding in different orientations (
      • Kallio P.
      • Patrikainen P.
      • Suomela J.-P.
      • Mäntsälä P.
      • Metsä-Ketelä M.
      • Niemi J.
      Flavoprotein hydroxylase PgaE catalyzes two consecutive oxygen-dependent tailoring reactions in angucycline biosynthesis.
      ,
      • Koskiniemi H.
      • Metsä-Ketelä M.
      • Dobritzsch D.
      • Kallio P.
      • Korhonen H.
      • Mäntsälä P.
      • Schneider G.
      • Niemi J.
      Crystal structures of two aromatic hydroxylases involved in the early tailoring steps of angucycline biosynthesis.
      ). We have previously demonstrated that in the absence of the C5-hydroxylase UbiI in E. coli, the FMO UbiF responsible for the C6-hydroxylation is able to support C5-hydroxylation, albeit not efficiently (
      • Hajj Chehade M.
      • Loiseau L.
      • Lombard M.
      • Pecqueur L.
      • Ismail A.
      • Smadja M.
      • Golinelli-Pimpaneau B.
      • Mellot-Draznieks C.
      • Hamelin O.
      • Aussel L.
      • Kieffer-Jaquinod S.
      • Labessan N.
      • Barras F.
      • Fontecave M.
      • Pierrel F.
      ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation.
      ). This result implies that the active site of UbiF accommodates two closely related substrates in different orientations compatible with hydroxylation at C5 and C6. Similarly, our results with Coq6 support that HHB and HAB, the substrates of the first hydroxylation at C5 must adopt a different conformation than HHAB, the substrate of the second hydroxylation at C4, to place the respective carbon atoms and the FAD-hydroperoxo in positions compatible with catalysis. In fact, Coq6 L382E and Coq6 M469X retained the capacity to hydroxylate at C5 but mostly lost the ability to hydroxylate C4 because deamination did not occur efficiently in these mutants (Fig. 4). Leu-382 lies within the substrate access channel, which may accommodate the hexaprenyl tail of the substrate as supported by our combined in silico and in vivo mutagenesis studies.6 The C-terminal part of Coq6, truncated in the M469X mutant, is in close proximity to this channel (Fig. 4B). Thus, we believe that the L382E and M469X mutations may affect differently the positioning of the diverse substrates. The position of HAB and HHB in the active site of these Coq6 mutants would still be compatible with C5-hydroxylation, whereas the position of HHAB would be perturbed and inadequate for hydroxylation at C4. Alternatively, the mutations may accelerate the release of HHAB from the active site and therefore preclude the second hydroxylation at C4. In either case, enzymes downstream of Coq6 convert HHAB into IDMQ6H2 (Fig. 1), which accumulates in the aforementioned Coq6 mutants (Fig. 4E). Actually, sensitive MS-MS detection revealed that IDMQ6 is also formed in minute amounts by WT S. cerevisiae cells cultivated in the presence of pABA (
      • Marbois B.
      • Xie L.X.
      • Choi S.
      • Hirano K.
      • Hyman K.
      • Clarke C.F.
      para-Aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae.
      ), showing that the C4-deamination reaction is not always completed even by a WT Coq6 enzyme. Similarly, IDDMQ6 was recently detected in the yeast coq5-5 mutant grown in the presence of pABA (
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ), although DDMQ6 is clearly the most abundant product in these cells (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ,
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ). There is no evidence that Coq6 may convert IDDMQ6 into DDMQ6 or IDMQ6 into DMQ6 and thus these late-stage C4-aminated compounds may actually represent dead end products of the Q biosynthetic pathway.

       Impact of coq9 Deletion on the C4-deamination Reaction

      In a recent study, Coq9 was proposed to control the deamination reaction (
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ). The results obtained in this study are compatible with our results that point to an indirect role of Coq9 in the C4-deamination reaction mediated by Coq6. Cells lacking Coq9 cultivated in the presence of pABA form 4-AP6 and IDMQ6 (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ,
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ). Accumulation of the former results from a C5-hydroxylation defect, whereas the latter demonstrates that Coq6 still catalyzes the C5-hydroxylation to some extent, whereas the C4-deamination is impaired (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ,
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ). We observed that Coq9 is indispensable to the C5-hydroxylase activity of Coq6 M469X (Fig. 5), which reveals that the C-terminal region of Coq6 is more specifically impacted by the absence of Coq9. Because the C-terminal region of Coq6 is important for the C4-deamination but quite dispensable for the C5-hydroxylation (Fig. 4E), we propose that the partial C5-hydroxylation defect and the profound C4-deamination deficit observed in Coq9-deficient cells (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ,
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ) result from a perturbation of the C-terminal part of Coq6. This hypothesis also accounts for the increased accumulation of IDDMQ6 in coq5–5 Δcoq9 cells (
      • He C.H.
      • Black D.S.
      • Nguyen T.P.
      • Wang C.
      • Srinivasan C.
      • Clarke C.F.
      Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.
      ). Our model of Coq6 shows that its C-terminal region is exposed at the protein surface (Fig. 4A), raising the possibility of an interaction with Coq9 via this domain. A direct interaction between Coq6 and Coq9 has yet to be proven, however, both proteins belong to the CoQ-synthome because Coq9 is co-immunoprecipitated and co-purified with Coq4, Coq5, Coq6, and Coq7 (
      • Allan C.M.
      • Awad A.M.
      • Johnson J.S.
      • Shirasaki D.I.
      • Wang C.
      • Blaby-Haas C.E.
      • Merchant S.S.
      • Loo J.A.
      • Clarke C.F.
      Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae.
      ,
      • Hsieh E.J.
      • Gin P.
      • Gulmezian M.
      • Tran U.C.
      • Saiki R.
      • Marbois B.N.
      • Clarke C.F.
      Saccharomyces cerevisiae Coq9 polypeptide is a subunit of the mitochondrial coenzyme Q biosynthetic complex.
      ). Recently, a direct interaction between human Coq9 and Coq7 proteins was demonstrated (
      • Lohman D.C.
      • Forouhar F.
      • Beebe E.T.
      • Stefely M.S.
      • Minogue C.E.
      • Ulbrich A.
      • Stefely J.A.
      • Sukumar S.
      • Luna-Sánchez M.
      • Jochem A.
      • Lew S.
      • Seetharaman J.
      • Xiao R.
      • Wang H.
      • Westphall M.S.
      • Wrobel R.L.
      • Everett J.K.
      • Mitchell J.C.
      • López L.C.
      • Coon J.J.
      • Tong L.
      • Pagliarini D.J.
      Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis.
      ). A deficiency of Coq9 in mammals seems to impact only Coq7 but not Coq6 unlike in S. cerevisiae (
      • Xie L.X.
      • Ozeir M.
      • Tang J.Y.
      • Chen J.Y.
      • Jaquinod S.K.
      • Fontecave M.
      • Clarke C.F.
      • Pierrel F.
      Over-expression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.
      ). Indeed, the only biosynthetic intermediate detected in tissues of the Coq9 R239X knock-in mouse was DMQ9, supporting a defect in Coq7 activity (
      • García-Corzo L.
      • Luna-Sánchez M.
      • Doerrier C.
      • García J.A.
      • Guarás A.
      • Acín-Pérez R.
      • Bullejos-Peregrín J.
      • López A.
      • Escames G.
      • Enríquez J.A.
      • Acuña-Castroviejo D.
      • López L.C.
      Dysfunctional Coq9 protein causes predominant encephalomyopathy associated with CoQ deficiency.
      ) but a normal activity of Coq6. The likely independence of human Coq6 with regard to Coq9 is in line with our results showing that the C5-hydroxylase activity of huCoq6 expressed in S. cerevisiae is actually hampered by the presence of Coq9 (Fig. 6). Notably, the absence of Coq9 also allowed huCoq6 to promote the C4-deamination reaction when expressed in S. cerevisiae (Fig. 6). This result definitely proves that Coq9 is not directly implicated in the C4-deamination and collectively, our results support that the C4-deamination defect observed in Δcoq9 cells is actually caused by perturbations of Coq6 structure.

       Conservation of the Capacity to Use pABA as a Substrate for Q Biosynthesis

      The respective contribution of endogenous 4-HB and pABA to Q biosynthesis is still unclear, although the predominant accumulation of 4-HP6 over 4-AP6 in Δcoq6 + pCOQ8 cells suggests that endogenous 4-HB is preferably used over pABA in these growth conditions (
      • Ozeir M.
      • Mühlenhoff U.
      • Webert H.
      • Lill R.
      • Fontecave M.
      • Pierrel F.
      Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
      ). pABA, is also a precursor in folate metabolism and is produced from chorismate by the action of Abz1, Abz2 (
      • Botet J.
      • Mateos L.
      • Revuelta J.L.
      • Santos M.A.
      A chemogenomic screening of sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers ABZ2, the gene encoding a fungal aminodeoxychorismate lyase.
      ), although 4-HB is proposed to originate from chorismate as well, but in an unidentified chain of reactions (
      • Clarke C.F.
      New advances in coenzyme Q biosynthesis.
      ). The cellular abundance of 4-HB and pABA, and therefore their contribution to Q biosynthesis, is likely to vary in S. cerevisiae depending on growth conditions (
      • Marbois B.
      • Xie L.X.
      • Choi S.
      • Hirano K.
      • Hyman K.
      • Clarke C.F.
      para-Aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae.
      ). Conceivably, other organisms than S. cerevisiae may use pABA as a precursor of Q if their C5-hydroxylase is able to function like Coq6. Recently, it was shown that Arabidopsis does not incorporate pABA into Q, however, it was not established whether pABA is prenylated or not in this organism (
      • Block A.
      • Widhalm J.R.
      • Fatihi A.
      • Cahoon R.E.
      • Wamboldt Y.
      • Elowsky C.
      • Mackenzie S.A.
      • Cahoon E.B.
      • Chapple C.
      • Dudareva N.
      • Basset G.J.
      The origin and biosynthesis of the benzenoid moiety of ubiquinone (coenzyme Q) in Arabidopsis.
      ). In E. coli, pABA yields several octaprenyl C4-aminated compounds but no Q8 (
      • Xie L.X.
      • Williams K.J.
      • He C.H.
      • Weng E.
      • Khong S.
      • Rose T.E.
      • Kwon O.
      • Bensinger S.J.
      • Marbois B.N.
      • Clarke C.F.
      Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis.
      ), implying that the C5-hydroxylase UbiI is not capable of catalyzing the C4-deamination. The potential C4-deamination capacity of a given Coq6 homolog is likely linked to the positioning of carbon C4 of HHAB with regard to the FAD as discussed in the case of L382E and M469X mutants. Such fine structural details will only be revealed by a crystal structure of the Coq6 protein, a task that is yet to be achieved.

      Author Contributions

      F. P. conceived and coordinated the study and wrote the manuscript. F. P., M. O., L. P., A. I., and C. M. D performed and analyzed the experiments. M. F. analyzed data and edited the paper. All authors reviewed the manuscript and approved the final version.

      Acknowledgments

      We thank Dr. Cathy Clarke (UCLA) for the Coq6 antibody and the Δcoq6 strains, Dr. Jean-Marc Latour (LCBM, Grenoble) for access to 18O2, and Dr. Leonardo Salviati (University of Padova) for the plasmids encoding Coq6 mutants. We thank Dr. Murielle Lombard (LCPB, Paris), Dr. Etienne Mulliez, Dr. Stéphane Menage, Dr. Pavel Sindelar (LCBM, Grenoble), and Dr. Patricia Renesto (LAPM, Grenoble) for insightful discussions.

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