Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis

Coenzyme Q (Q or ubiquinone) is a polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain and in the plasma membrane of Escherichia coli (1, 2). The hydroquinone or reduced form [coenzyme QH2 or ubiquinol (QH2)] functions as a chain-terminating lipid antioxidant and as a coantioxidant to recycle vitamin E (3). Q is also involved in many other metabolic processes, including fatty acid β-oxidation, sulfide oxidization, disulfide bond formation, and pyrimidine metabolism (4–7). Q is composed of a fully substituted benzoquinone ring that is attached to a polyisoprenyl tail with a variable number of isoprenyl units (six for Saccharomyces cerevisiae, eight for E. coli, nine for mouse, and ten for human, hence Q10). Most cells rely on de novo synthesis for sufficient amounts of Q, although brown adipose tissue was recently discovered to rely on uptake of exogenously supplied Q (8). In baker’s yeast, S. cerevisiae, at least 13 gene products, Coq1-Coq11, Arh1, and Yah1 (9–14) are essential for Q biosynthesis. The Coq1 polypeptide synthesizes the polyisoprenyl tail and Coq2 attaches the tail to the aromatic ring (Fig. 1). The other Coq polypeptides catalyze ring modifications including O-methylation (Coq3), C-methylation (Coq5), hydroxylation (Coq6 and Coq7), and the function of Coq6 requires ferredoxin (Yah1) and ferredoxin reductase (Arh1) (9). The roles of Coq4, Coq8, Coq9, Coq10, and Coq11 have not yet been determined, although they are all required for efficient yeast Q biosynthesis. Schemes of Q biosynthesis generally depict 4-hydroxybenzoic acid (4HB) as the biosynthetic aromatic ring precursor of Q (4). 4HB is considered to derive from chorismate in yeast and from phenylalanine or tyrosine in animal cells (15–17). Yeast can also use para-aminobenzoic acid (pABA) as an alternate ring precursor in the biosynthesis of Q (9, 18). This finding was surprising because pABA is a well-known precursor of folate, which is synthesized de novo by many microorganisms and folate is a vitamin for humans. A biosynthetic scheme was reported recently including proposed steps for the conversion of pABA to Q6 in S. cerevisiae (19). Fig. 1. Schemes of Q biosynthesis in S. cerevisiae, other eukaryotes, and E. coli. In S. cerevisiae, Coq1 synthesizes the hexaprenyl diphosphate tail, and Coq2 adds the hexaprenyl tail (denoted as “R”) to either 4HB or to pABA, forming 3-hexaprenyl-4HB ... The biosynthesis of Q8 in E. coli requires IspB (which synthesizes the octaprenyl diphosphate tail precursor) (20) and 11 Ubi polypeptides (UbiA–UbiJ and UbiX; Fig. 1) (21). UbiC carries out the first committed step in the biosynthesis of Q8, the conversion of chorismate to 4HB (22). UbiA adds the octaprenyl tail to the 4HB ring, followed by the decarboxylation catalyzed by UbiD and UbiX. UbiI adds the first hydroxyl group at the C5 position, followed by O-methylation catalyzed by UbiG, the homolog of yeast Coq3. Additional ring modifications catalyzed by UbiH, UbiE, UbiF, and UbiG generate the final product of Q8. UbiB, an atypical protein kinase similar to Coq8, and UbiJ play essential, but unknown, functions in E. coli Q8 biosynthesis (21). Recently, Block et al. (15) identified para-coumarate (p-coumarate) as a ring precursor of Q biosynthesis in Arabidopsis thaliana. Arabidopsis converts phenylalanine to p-coumarate in the cytosol, and following transport into peroxisome, p-coumarate is ligated to CoA and the three-carbon side chain is shortened via peroxisomal β-oxidation (15). Plant peroxisomes appear to contain thiolases and CoA thioesterases that can ultimately produce 4HB from 4-hydroxybenzoyl-CoA (15). Tyrosine can also supply the ring of Q in Arabidopsis; but this must occur via a nonintersecting pathway, because Arabidopsis mutants unable to utilize phenylalanine still utilized tyrosine as a ring precursor of Q (15). Animal cells are able to hydroxylate phenylalanine to form tyrosine, and it is presumed that conversion of tyrosine to 4HB occurs via its metabolism to p-coumarate (16, 23). However, the enzymes involved in 4HB biosynthesis in either yeast or animal cells have not been identified. The in vivo metabolism of potential ring precursors labeled with the stable isotope 13C can be determined with high sensitivity and specificity with reverse phase (RP)-HPLC-MS/MS identification and quantification. Using this approach, Block et al. (15) showed that Arabidopsis was not able to incorporate 13C6-pABA into Q. Here, we have made use of 13C6-ring-labeled forms of pABA and p-coumarate to track their metabolic fate as potential Q biosynthetic precursors in E. coli, S. cerevisiae, and animal cells. Due to its structural similarity with p-coumarate, 13C6-resveratrol was also tested as a ring precursor in Q biosynthesis. In this study, we found that human and E. coli cells do not utilize pABA as an aromatic ring precursor in the synthesis of Q, while resveratrol and p-coumarate serve as ring precursors of Q in E. coli, S. cerevisiae, and human cells.