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Analysis and characterization of the biosynthetic pathway of Coenzyme Q in eukaryotes, and the role of ring precursors and key intermediates

  • Author(s): Nag, Anish
  • Advisor(s): Clarke, Catherine F
  • et al.
Abstract

Coenzyme Q (known by various names that include ubiquinone, CoQ or simply Q) is a crucial redox-active lipid that consists of a fully substituted benzenoid head group and a polyisoprenoid tail. The benzenoid head group moiety that resembles a quinone, can undergo reversible redox reactions interconverting from the fully oxidized quinone through a radical semi-quinone intermediate to the fully reduced quinol. This structural feature aids in the essential role that Q plays in cellular respiration, wherein it transports electrons from NADH and succinate to cytochrome c (Respiratory complex I and II to III respectively in eukaryotes). Q also acts as a lipid soluble chain terminating anti-oxidant. Thus, complete lack of Q is embryonically fatal and sufficient de novo Q biosynthesis is crucial for proper health maintenance in humans. Q deficiency has been directly or indirectly linked to a wide spectrum of health disorders in humans, including kidney disease, neurodegenerative diseases, cerebellar ataxia, and cardiovascular complications. Additionally, decreased Q levels have been linked to aging. Current therapeutic strategies to treat Q deficiency related complications involve direct oral supplementation of Q, which has its challenges due to the hydrophobicity and low bio-availability of Q. Therefore, our research is aimed at characterizing the biosynthesis and metabolism of Q in living cells, thereby potentially leading the way to novel therapeutic techniques.

Saccharomyces cerevisiae (yeast) serves as a highly useful model for research on Q, due to its widely studied molecular genetics and its close homology to human Q biosynthesis, metabolism and function. Q biosynthesis in S. cerevisiae (which makes Q6 with six isoprene units, versus humans whose Q10 has ten isoprene units) takes place in the mitochondria. Chapter 1 highlights the currently known Q biosynthetic steps along with phenotypes observed from deletion and malfunction of Q biosynthesis in yeast and humans. The primary precursor molecule that is utilized by eukaryotes to biosynthesize Q is 4-hydroxybenzoic acid (4HB). The latter is in turn immediately preceded by 4-hydroxybenzaldehyde (4HBz) in the Q biosynthetic pathway. Fourteen known proteins that localize in the mitochondria are responsible for catalyzing different steps in this process—Coq1-Coq11, Yah1 (ferredoxin), Arh1 (ferredoxin reductase), and Hfd1 (aldehyde dehydrogenase). In yeast 4HBz is biosynthesized from the precursor amino acid Tyrosine (Tyr). However, this pathway lacks proper characterization and only the deaminases Aro8 and Aro9 and the aldehyde dehydrogenase Hfd1 have been identified. The human homolog of Hfd1 is ALDH3A1 which can serve as a potential target when attempting to screen for Q deficiency in humans.

Chapter 2 investigates the role of key proteins and intermediates in the biosynthesis of Q in yeast. In addition to 4HB yeast is also capable of utilizing p-aminobenzoic acid (pABA) as a Q ring precursor. However, the exact steps by which the two pathways converge was not fully characterized. It was postulated that the deamination followed by hydroxylation of the pABA phenyl ring occurs via Schiff base chemistry. Additionally, high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis of yeast mutants with deletions in selected Coq genes, showed accumulation of Q intermediates. These intermediates included 3-hexaprenyl-4-hydroxyphenol (4-HP), 3-hexaprenyl-4-aminophenol (4-AP), demethyl demethoxy Q6 (DDMQ6), imino demethyl demethoxy Q6 (IDDMQ6), demethoxy Q6 (DMQ6), and imino demethoxy Q6 (IDMQ6). In order to test the hypothesis of the Schiff base chemistry responsible for Q biosynthesis from pABA, and to investigate whether the above mentioned intermediates are actual productive Q intermediates or just dead-end intermediates, farnesylated analogs (wherein the hexaprenyl tail of Q intermediates is changed to a farnesyl tail consisting of three isoprene units) of 4-HP, DDMQ6 and DMQ6 along with the reduced intermediate IDDMQ6H2, were chemically synthesized. Thus 2-farnesyl-4-dyroxyphenol (4-HFP), demethyl demethoxy Q3 (DDMQ3), demethoxy Q3 (DMQ3) and reduced imino demethyl demethoxy Q3 (IDDMQ3H2) were correspondingly obtained. These intermediates were fed to yeast in biochemical feeding assays and their corresponding potential transformation to Q3 was analyzed via HPLC-MS/MS studies. DMQ3 showed ready conversion to Q3. However, DDMQ3 showed very limited Q3 generation, whereas, 4-HFP and IDDMQ3H2 failed to show detectable levels of Q3. Thus the role of DMQ6 as a Q biosynthetic intermediate was further elucidated.

It was demonstrated by Dr. Fabien Pierrel’s research group that the Schiff base chemistry hypothesis for the convergence of the pABA and 4HB pathways leading to Q biosynthesis, was incorrect, and the deamination of the ring of pABA occurs further upstream than formerly postulated. Therefore efforts to further synthesize and investigate farnesylated analogs of Q intermediates were postponed and instead investigations were carried out to test the role of ring precursors in addition to 4HB and pABA in Q biosynthesis. Chapter 3 gives details of the studies conducted on selected alternate Q ring precursors, and the discovery of kaempferol (a plant derived flavonol), as a novel Q ring precursor in mammalian cells. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4′ of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney.

In addition to kaempferol, other phenolic molecules were previously shown to act as Q ring precursors in yeast and mammalian cells. This included p-coumaric acid. Chapter 4 reports a detailed investigation into the role of p-coumaric acid as a Q ring precursor in yeast. Stable isotope labeled [13C6-ring]-p-coumaric acid was chemically synthesized. This was tested on BY4741 and W303 genetic backgrounds of wild type (WT) yeast to analyze corresponding [13C6-ring]-Q levels via HPLC-MS/MS. Different growth media conditions and times of incubation were utilized in order to fully assess the role of p-coumaric acid as an alternate Q ring precursor. It was discovered that the W303 genetic background of yeast has a much higher efficiency of p-coumaric acid uptake and subsequent conversion to Q. Furthmore, attempts were made to test the pathway by which p-coumaric acid is biosynthesized to Q. It was postulated that this occurs via intermediary biosynthesis of the former to 4HB. To investigate this possibility the Hfd1 gene was knocked out in the W303 genetic background, and the corresponding hfd1 null strain was assayed with [13C6-ring]-p-coumaric acid.

Chapter 5 provides insight and perspectives into projects being currently pursued and potential experiments to be conducted in the future. In particular, we are probing further into the role of kaempferol as a Q ring precursor in mammalian cells. It was hypothesized that the B ring of kaempferol underwent cleavage from the rest of the molecule and was utilized to generate the ring of Q in mammalian cells. This hypothesis was further strengthened when Dr. Gilles Basset was able to confirm the utilization of the B ring of kaempferol to generate Q in Arabidopsis thaliana. Moreover, Dr. Bassett showed that this occurs through a peroxidative cleavage mechanism, whereby the B ring of kaempferol is converted to 4HB, which in turn is used to generate Q. Attempts are being made to explore a similar potential peroxidative mechanism occurring in mammalian cells. An in vitro peroxidation assay similar to the one used by Dr. Bassett is in the process of being set up on mammalian cell extracts incubated with kaempferol. Methods have been generated to detect 4HB (synthesized by kaempferol peroxidation in the cell extracts) by HPLC-MS/MS via a derivatization strategy. In addition, mouse kidney cells grown in presence of kaempferol with the B ring selectively labeled with stable 13C isotope (generated by Dr. Bassett), have shown production of 13C stable isotope ring labeled Q.

Finally, the Appendix contains two additional publications. The first explores alternative splicing in yeast and the role it plays in Q biosynthesis. PTC7 encodes the phosphatase responsible for the dephosphorylation of Coq7 undergoes alternative splicing, which is rare in yeast. The study also implicated SNF2 as the gene that is responsible for this alternative splicing event and showed that deletion of SNF2 leads to increased Q levels in yeast. The second publication explores the rescue of the clinical phenotypes associated with Coq6 deletion in mice by supplementation with 2,4-dihydroxybenzoic acid. In particular it was shown that steroid resistance nephrotic syndrome which develops in mice with Coq6 deletion, can be ameliorated by treatment with 2,4-dihydroxybenzoic acid.

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