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The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway

Abstract

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1,2,3,4 and cancers with HPDL overexpression5.

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Fig. 1: Analysis of the oxy-metabolome identifies 4-HMA as a highly 18O-labelled metabolite in human cells.
Fig. 2: 4-HMA is derived from tyrosine and synthesized by HPDL (4-hydroxyphenylpyruvate dioxygenase-like) in human cells.
Fig. 3: HPDL and 4-HMA participate in the human CoQ10 headgroup biosynthesis pathway.

Data availability

TLCV2-sgHPDL and coHPDL expression plasmids are available from Addgene (IDs 174128, 174129, 174130, 174131, 174165 and 174166). All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. 1.

    Morgan, N. V. et al. Evidence that autosomal recessive spastic cerebral palsy-1 (CPSQ1) is caused by a missense variant in HPDL. Brain Commun. 3, fcab002 (2021).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  2. 2.

    Husain, R. A. et al. Bi-allelic HPDL variants cause a neurodegenerative disease ranging from neonatal encephalopathy to adolescent-onset spastic paraplegia. Am. J. Hum. Genet. 107, 364–373 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Ghosh, S. G. et al. Biallelic variants in HPDL, encoding 4-hydroxyphenylpyruvate dioxygenase-like protein, lead to an infantile neurodegenerative condition. Genet. Med. 23, 524–533 (2021).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Wiessner, M. et al. Biallelic variants in HPDL cause pure and complicated hereditary spastic paraplegia. Brain 144, 1422–1434 (2021).

    PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Ye, X. et al. 4-hydroxyphenylpyruvate dioxygenase-like protein promotes pancreatic cancer cell progression and is associated with glutamine-mediated redox balance. Front. Oncol. 10, 3074 (2021).

    Google Scholar 

  6. 6.

    Tang, D., Kang, R., Berghe, T. V., Vandenabeele, P. & Kroemer, G. The molecular machinery of regulated cell death. Cell Res. 29, 347–364 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Hirsila, M., Koivunen, P., Gunzler, V., Kivirikko, K. I. & Myllyharju, J. Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor. J. Biol. Chem. 278, 30772–30780 (2003).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  8. 8.

    Masson, N. et al. Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants. Science 365, 65–69 (2019).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Laukka, T. et al. Fumarate and succinate regulate expression of hypoxia-inducible genes via TET enzymes. J. Biol. Chem. 291, 4256–4265 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Moran, G. R. 4-Hydroxyphenylpyruvate dioxygenase. Arch. Biochem. Biophys. 433, 117–128 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Ast, T. & Mootha, V. K. Oxygen and mammalian cell culture: are we repeating the experiment of Dr. Ox? Nature Metabolism 1, 858–860 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Deshpande, A. R., Wagenpfeil, K., Pochapsky, T. C., Petsko, G. A. & Ringe, D. Metal-dependent function of a mammalian acireductone dioxygenase. Biochemistry 55, 1398–1407 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Drazic, A. & Winter, J. The physiological role of reversible methionine oxidation. Biochim. Biophys. Acta 1844, 1367–1382 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Choroba, O. W., Williams, D. H. & Spencer, J. B. Biosynthesis of the vancomycin group of antibiotics:  involvement of an unusual dioxygenase in the pathway to (S)-4-hydroxyphenylglycine. JACS 122, 5389–5390 (2000).

    CAS  Article  Google Scholar 

  15. 15.

    Hubbard, B. K., Thomas, M. G. & Walsh, C. T. Biosynthesis of l-p-hydroxyphenylglycine, a non-proteinogenic amino acid constituent of peptide antibiotics. Chem. Biol. 7, 931–942 (2000).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  16. 16.

    Lemberger, L., Klutch, A. & Kuntzman, R. The metabolism of tyramine in rabbits. J. Pharmacol. Exp. Ther. 153, 183 (1966).

    CAS  Google Scholar 

  17. 17.

    Lichter-Konecki, U., Hipke, C. M. & Konecki, D. S. Human phenylalanine hydroxylase gene expression in kidney and other nonhepatic tissues. Mol. Genet. Metab. 67, 308–316 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Gunsior, M., Ravel, J., Challis, G. L. & Townsend, C. A. Engineering p-hydroxyphenylpyruvate dioxygenase to a p-hydroxymandelate synthase and evidence for the proposed benzene oxide intermediate in homogentisate formation. Biochemistry 43, 663–674 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    O’Hare, H. M., Huang, F., Holding, A., Choroba, O. W. & Spencer, J. B. Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution. FEBS Lett. 580, 3445–3450 (2006).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  20. 20.

    Gunsior, M. et al. The biosynthetic gene cluster for a monocyclic β-lactam antibiotic, nocardicin A. Chem. Biol. 11, 927–938 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Bhat, S. G. & Vaidyanathan, C. S. Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa. J. Bacteriol. 127, 1108–1118 (1976).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Stefely, J. A. & Pagliarini, D. J. Biochemistry of mitochondrial coenzyme Q biosynthesis. Trends Biochem. Sci. 42, 824–843 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Lu, T.-T., Lee, S. J., Apfel, U.-P. & Lippard, S. J. Aging-associated enzyme human clock-1: substrate-mediated reduction of the diiron center for 5-demethoxyubiquinone hydroxylation. Biochemistry 52, 2236–2244 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Wang, Y. et al. The anti-neurodegeneration drug clioquinol inhibits the aging-associated protein CLK-1. J. Biol. Chem. 284, 314–323 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Payet, L.-A. et al. Mechanistic details of early steps in coenzyme Q biosynthesis pathway in yeast. Cell Chem. Biol. 23, 1241–1250 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Stefely, J. A. et al. Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling. Nat. Biotechnol. 34, 1191–1197 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Valera, M. J. et al. The mandelate pathway, an alternative to the phenylalanine ammonia lyase pathway for the synthesis of benzenoids in ascomycete yeasts. Appl. Environ. Microbiol. 86, e00701–20 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Kaymak, I. et al. Mevalonate pathway provides ubiquinone to maintain pyrimidine synthesis and survival in p53-deficient cancer cells exposed to metabolic stress. Cancer Res. 80, 189–203 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Kapalczynska, M. et al. 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch. Med. Sci. 14, 910–919 (2018).

    PubMed  PubMed Central  Google Scholar 

  30. 30.

    Doimo, M. et al. Genetics of coenzyme q10 deficiency. Mol. Syndromol. 5, 156–162 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Martínez-Reyes, I. et al. Mitochondrial ubiquinol oxidation is necessary for tumour growth. Nature 585, 288–292 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. 32.

    Xie, L. X. et al. Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis. J. Lipid Res. 56, 909–919 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Fernández-del-Río, L. et al. Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor. Free Radical Biol. Med. 110, 176–187 (2017).

    Article  CAS  Google Scholar 

  34. 34.

    Booth, A. N. et al. Urinary phenolic acid metabolites of tyrosine. J. Biol. Chem. 235, 2649–2652 (1960).

    CAS  Article  Google Scholar 

  35. 35.

    Hartl, J., Kiefer, P., Meyer, F. & Vorholt, J. A. Longevity of major coenzymes allows minimal de novo synthesis in microorganisms. Nat Microbiol 2, 17073 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Meyers, R. M. et al. Computational correction of copy number effect improves specificity of CRISPRCas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779–1784 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Uphoff, C. C. & Drexler, H. G. Detecting mycoplasma contamination in cell cultures by polymerase chain reaction. Methods Mol. Biol. 731, 93–103 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Parker, S. J. et al. LKB1 promotes metabolic flexibility in response to energy stress. Metab. Eng. 43, 208–217 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Wenig, P. & Odermatt, J. OpenChrom: a cross-platform open source software for the mass spectrometric analysis of chromatographic data. BMC Bioinf. 11, 405 (2010).

    Article  CAS  Google Scholar 

  40. 40.

    Fernandez, C. A., Des Rosiers, C., Previs, S. F., David, F. & Brunengraber, H. Correction of 13C mass isotopomer distributions for natural stable isotope abundance. J. Mass Spectrom. 31, 255–262 (1996).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41.

    Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 6, pl1 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. 42.

    Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).

    PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D. M. Sabatini for advice and for critical reading of the manuscript; R. L. Possemato and his laboratory for use of an incubator with physiologic oxygen (3% O2); B. G. Neel for support at the inception of this project; and members of the Kimmelman and Pacold laboratories for their help and suggestions. We acknowledge the NYU Langone Health Experimental Pathology Research Laboratory, Microscopy Laboratory and Metabolomics Core Resource Laboratory for their help in acquiring the data presented. These shared resources are partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. R.S.B. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2348-18). Q.S. was supported by the Wallonie-Bruxelles International (WBI) fellowship. D.E.B. is supported by a Ruth L. Kirschstein Institutional National Research Service Award, T32 CA009161 (Levy), and the NCI F99/K00 award (F99 CA245822). K.Y. was supported by an Uehara Memorial Foundation Research Fellowship. A.C.K. is supported by National Cancer Institute Grants R01CA157490, R01CA188048, P01CA117969, R35CA232124; ACS Research Scholar Grant RSG-13-298-01-TBG; NIH grant R01GM095567; the Lustgarten Foundation, and Stand Up to Cancer (SU2C), a division of the Entertainment Industry Foundation. SU2C is administered by the AACR. M.E.P. is a Damon Runyon-Rachleff Innovation Awardee supported in part by the Damon Runyon Cancer Research Foundation (DRR 63-20), and is supported by a Mary Kay Foundation Cancer Research Grant (017-32), the Shifrin-Myers Breast Cancer Discovery Fund at NYU, a V Foundation V Scholar Grant funded by the Hearst Foundation (V2017-004), an NCI K22 Career Transition Award (K22CA212059), the Tara Miller Melanoma Foundation – MRA Young Investigator Award (668365), the Harry J. Lloyd Trust, and laboratory-directed research funding from Max Raskin.

Author information

Affiliations

Authors

Contributions

R.S.B. conceived, planned and guided the research, designed and performed all the experiments with assistance as described, analysed and interpreted the data, and wrote the manuscript. E.S.K. contributed to, and analysed the data for, the labelling experiments, in vitro growth measurements and in vivo tumour studies. Q.S. provided biochemistry and chemistry expertise, and contributed to in vivo tumour studies. D.E.B., K.Y., A.S.W.S., G.S. and Q.S. performed mouse surgeries for orthotopic xenografts in the pancreas, and G.S. obtained tumour-response data. D.R.J. provided expertise on mass spectrometry and fragmentation. A.C.K. provided conceptual advice for the project. M.E.P. carried out gaseous labelling experiments, provided expertise on mass spectrometry, interpreted the data, supervised the project and wrote the manuscript. All authors critically analysed data, and edited and approved the manuscript.

Corresponding author

Correspondence to Michael E. Pacold.

Ethics declarations

Competing interests

A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to KRAS-regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach, and the autophagic control of iron metabolism. A.C.K is on the SAB of Rafael/Cornerstone Pharmaceuticals. A.C.K has been a consultant for Diciphera Pharmaceuticals. M.E.P. has options in Raze Therapeutics, is the recipient of travel funds from Thermo Fisher Scientific and consulted for aMoon Ventures. R.S.B., Q.S., and M.E.P. are co-inventors on a patent filing on aspects of CoQ10 metabolism. The other authors declare no competing interests.

Additional information

Peer review information Nature thanks Costas Lyssiotis, Joshua Rabinowitz, Jared Rutter and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 A robust method for 18O2 labelling of human cells.

a, Schematic of 18O2 labelling. A closed system chamber is flushed multiple times with N2 to remove 16O2. A gas mixture containing 18O2 and CO2 is pulsed into the closed chamber to reach the desired oxygen concentration. At the assay endpoint, the chamber is opened, cells are extracted, and metabolites separated and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). b, Oxygen measurements after N2 flush, followed with or without pulses of O2:CO2 gas mixture in the closed chamber containing tissue culture plates and media (n = 3 technical replicates each). ce, Oxygen percentage of O2-labelling experiments performed at 3% (c), 1% (d), and 0.2% (e) 16O2 or 18O2. (n = 2 technical replicates each). f, Cells were treated with several concentrations of MG132, DFO (an iron chelator), IOX1 (a dioxygenase inhibitor), or in combination at 5% O2 for 24 h. Immunoblots of HIF1α with ERK2 as a loading control. g, Immunoprecipitation of HIF1α to determine its hydroxylation (P564-OH) levels by the indicated inhibitors. Immunoblots of HIF1α and HIF1α P564-OH are shown, with ERK2 serving as a loading control. Experiments were performed once for optimization of drug concentrations (f, g). Graphs represent mean ± s.d. (ce).

Source data

Extended Data Fig. 2 18O2 labelling of human cells reveals the oxy-metabolome.

a, Schematic of the approach used to identify 18O-labelled features and metabolites that were labelled by 18O. n represents the number of features or metabolites identified in MIAPACA2 cells grown at 3% 18O2. See b for details. b, Summary of total and percentage of identified 18O-labelled features for each cell line and oxygen tension as described in a. c, Venn diagram demonstrating the overlap of unique 18O-labelled metabolite and features identified for each oxygen condition per cell line. d, Total number of unique dioxygenase-dependent, 18O-labelled metabolites and features identified in each cell line and condition. Features were categorized into predicted or not predicted/unknown 18O-labelled metabolites, based on known oxygen-dependent metabolic pathways, and sensitivity or insensitivity to IOX1 (dioxygenase inhibitor) treatment. e, Number of 18O-labelled metabolites detected in cells grown in 3%, 1%, and 0.2% 18O2 for 24 h in two sets of experiments. The overlap of the total number of detected 18O-labelled metabolites and features in both experimental sets are shown. f, Venn diagram representing the distribution of common and unique 18O-labelled metabolites identified in each cell line. g, List of the 46 unique 18O-labelled metabolites that were identified in f and categorized into known oxygen-dependent metabolic pathways. ** represents metabolites that have matching MS2 spectra, but need to be validated due to multiple metabolite isomers.

Source data

Extended Data Fig. 3 Fractional 18O labelling of metabolites and features identified in human cells by 18O2 labelling.

a, Heatmap representing the median fractional 18O labelling of the 46 metabolites and features in the indicated cell lines and oxygen tensions. “**” represents metabolites that have matching MS2 spectra, but need to be validated due to multiple metabolite isomers. The red arrow indicates a highly labelled unknown metabolite. b, Correlation matrixes demonstrating the Spearman rs value based on the fractional 18O labelling of the 46 metabolites and features across the indicated cell lines and oxygen tensions. c, Fractional 18O labelling of unknown feature (167.0339 in negative ion mode, elution time of 8.2 min) in MIAPACA2, A498, and SKNDZ cells grown in 3%, 1%, and 0.2% 18O2, and treated with vehicle or IOX1 (dioxygenase inhibitor) for 24 h (n = 3). di, Fractional 18O labelling of metabolites and features by 18O2 across multiple cell lines in response to different oxygen tensions, treated with or without IOX1 (dioxygenase inhibitor) for 24 h. 18O labelling of predicted (de), not predicted (fh), and unknown (i) metabolites or features are shown for the indicated cell line. ** represents metabolites that have matching MS2 spectra, but need to be validated due to multiple metabolite isomers. n = 3 biologically independent samples for each group and condition in all experiments. Graphs represent mean ± s.e.m. and were compared using one- (b–d) or two-way ANOVA (a, ef), followed by Tukey post hoc test (*P < 0.05, ^P < 0.01, %P < 0.005, #P< 0.0001).

Source data

Extended Data Fig. 4 Total levels of unlabelled and 18O-labelled metabolites identified in human cells.

a, Schematic of the carnitine biosynthesis pathway. Dioxygenases, TMLH (Trimethyllysine hydroxylase) and BBOX (butyrobetaine, 2-oxoglutarate dioxygenase), are shown in orange boxes, and 18O labelling is indicated in blue by arrows. bd, Total intracellular levels of unlabelled and 18O-labelled γ-butyrobetaine from cells grown in 3%, 1%, and 0.2% 18O2 with the indicated reagents for 24 h (n = 3). e, Schematic of the methionine salvage pathway. ADI1 (Acireductone dioxygenase 1), a dioxygenase, is shown in orange, and 18O labelling is indicated in blue with arrows. fh, Total intracellular levels of unlabelled and 18O-labelled methionine from cells grown in 3%, 1%, and 0.2% 18O2 with the indicated reagents for 24 h (n= 3). i, Schematic of methionine oxidation by 18O-labelled reactive oxygen species indicated by arrows. jm, Total intracellular levels of unlabelled and 18O-labelled methionine sulfoxide from cells grown in 3%, 1%, and 0.2% 18O2 with the indicated reagents for 24 h (n=3). n represents the number of biologically independent experiments for each group and condition. Graphs (mean ± s.e.m.) were compared using two-tailed Student t-test (cd, gh, km) or one-way (b, f j) ANOVA, followed by Tukey post hoc test (*P< 0.05, ^P< 0.01, %P< 0.005, #P< 0.0001).

Source data

Extended Data Fig. 5 Identification of 18O-labelled 4-HMA in human cells.

a, Tandem mass spectra (MS2) of homogentisate (HGA) standard, 4-hydroxymandelate (4-HMA) standard, unlabelled (167.0344 m/z) feature precursors, and the respective product fragments. Mass differences between the precursor and product ions reflect loss of one CO2. The red line indicates fragmentation of the precursor ion into the two product ions. The structure of the precursor and product ions are depicted on the left. b, MS2 of unlabelled (167.0344 m/z), +one 18O (169.0387 m/z), and +two 18O (171.0428 m/z) labelled 4-HMA precursors, and the respective product fragments. Mass differences between precursor and product ions reflects loss of unlabelled and +one 18O-labelled CO2. The red line indicates precursor ion fragmentation into two product ions. The structure and position of 18O-labelled (blue and arrow) 4-HMA are depicted on the left. cd, Total levels of unlabelled and 18O-labelled 4-HMA levels in A498 (c) and SKNDZ (d) cells grown in 3%, 1%, and 0.2% 18O2, and treated with or without IOX1 (dioxygenase inhibitor) for 24 h. n= 3 in biologically independent replicates for each group and condition. Graphs represent mean ± s.e.m. and were compared by two-way ANOVA (cd), followed by Bonferroni post hoc test (*P< 0.05, ^P< 0.01, %P< 0.005, #P< 0.0001).

Source data

Extended Data Fig. 6 4-HMA is a tyrosine-derived metabolite synthesized from tyrosine in human cells.

a, Schematic of known and proposed pathways involved in 4-HMA biosynthesis found in the literature. A. orientalis biosynthesizes 4-HMA from 4-hydroxyphenylpyruvate (4-HPPA), via hydroxymandelate synthase (HmaS), an Fe-dioxygenase. 4-HMA also has been proposed to be made from tyramine in rabbits by radioactive tracing studies. However, the proposed pathway was never formally demonstrated, as indicated by the dotted lines and box. b, Fractional labelling of Phe, Tyr, 4-HPPA, 4-HPLA, and 4-HMA from cells grown at 3% O2 with or without 13C9-Tyr or 13C6-Phe for 24 h (n= 5 for each group). c, Total intracellular levels of unlabelled and 13C-labelled Tyr, 4-HPPA, 4-HPLA, and 4-HMA from cells grown at 3%, 1%, and 0.2% O2 with 13C-Tyr for 24 h (n= 5 for each group). d, Total intracellular levels of unlabelled and 13C-labelled Tyr, 4-HPPA, 4-HPLA, and 4-HMA from cells grown in 13C-Tyr at 3% O2 with the indicated reagents for 24 h (n= 5 for each group). n represents the number of biologically independent replicates for each group and condition. Graphs represent mean ± s.e.m. and were compared by two-way ANOVA (bd), followed by Tukey post hoc test (*P< 0.05, ^P< 0.01, %P< 0.005, #P< 0.0001).

Source data

Extended Data Fig. 7 Human HPDL is an ortholog of A. orientalis HmaS.

a, b, Phylogenetic tree of HPD, HPDL, and HmaS cDNA (a) and protein (b) sequences across several model organisms. c, Protein sequence alignment of HPD, HPDL and HmaS. Catalytic histidines involved in coordinating the iron ion needed for activity are highlighted in red. Specific residues in Steptomyces avermitilis and Pseudomonas fluorescens HPD have been mutated in other studies, and the human equivalent mutations are as indicated; hydrophobic (blue), polar (green) amino acids and proline (yellow). The HPD P239T mutant decreases HGA production and generates oxopinone. The N241I/L mutation abolishes HGA production by HPD. The HPD S226A mutations blocks HGA production. However, the mutation in the equivalent site in HMS (S201A) does not affect the generation of 4-HMA. The F337V/L mutation in HPD decreases HGA synthesis and allows slight production of 4-HMA. d, Growth curve of MIAPACA2 cells with sgRNAs at 21% O2. (n= 3 technical replicates for each cell line, performed at least twice). e, Unlabelled and 13C8-labelled 4-HMA from PATU-8902 cells grown in 13C9-Tyr at 21% 16O2 for 24 h. (n=3). Immunoblots of HPDL levels from PATU-8902 cells expressing control and HPDL sgRNAs. ERK2 serves as a loading control. f, Unlabelled and 13C-labelled Tyr, 4-HPPA, 4-HPLA from MIAPACA2 cells were grown in 13C9-Tyr at 21% 16O2 for 24 h (from Fig. 2a). (n=5). g, Unlabelled and 13C-labelled Tyr, 4-HPPA, 4-HPLA from MIAPACA2 sgHPDL #3 cells were grown in 13C9-Tyr at 21% 16O2 for 24 h (from Fig. 2b). (n=5). n represents the number of biologically independent replicates for each group and condition, unless indicated (d). Graphs are represented as mean ± s.d. (d) or s.e.m. (eg) and were compared by two-tailed Student t-test (e), or two-way ANOVA (d, f, g), followed by Tukey post hoc test (*P< 0.05, ^P< 0.01, %P< 0.005, #P< 0.0001).

Source data

Extended Data Figure 8 Expression of HPDL affects 13C9-Tyr labelling of CoQ10.

a, Schematic of known and unknown components of the CoQ10 biosynthesis pathway in humans. R1 reflects the polyprenyl tail that is attached to 4-HB. b, Fractional labelling and total levels of CoQ10 from MIAPACA2 grown in unlabelled, 13C9-Tyr-, and 13C6-Phe-labelled media for 24 h at 3% 16O2. (n=5). c, Fractional labelling and total levels of CoQ10 from MIAPACA2 cells grown in 13C9-Tyr at 21%, 3% and 0.1% 16O2 for 24 h (n=5). d, Fractional labelling and total levels of CoQ10 from MIAPACA2 cells grown in 13C9-Tyr containing media, with the indicated compounds at 3% 16O2 for 24 h (n=5). e, Fractional labelling and total intracellular levels of unlabelled and 13C6-labelled CoQ10 from the indicated MIAPACA2 cells. Cells were grown in 13C9-Tyr at 21% 16O2 for 24 h (n=5). f, Fractional labelling of CoQ10 from indicated MIAPACA2 cells. Cells were grown in 13C9-Tyr at 21% 16O2 for 24 h. (n=5). g, Unlabelled and 13C6-labelled CoQ10 levels from PATU-8902 cells grown in 13C9-Tyr media (n=3). h, Extracellular concentrations of 13C8-labelled 4-HMA released from MIAPACA2 cells expressing control and HPDL sgRNAs. Cells were grown in 13C9-Tyr at 21% 16O2 for 24 h at low (LD) and high (HD) cell densities (n=3). Representative images of LD and HD cells are shown. i, The effect of cell density on total intracellular levels of unlabelled and 13C6-labelled CoQ10 from the indicated MIAPACA2 cells. Cells were grown in 13C9-Tyr at 21% 16O2 for 24 h at LD and HD (n=3 for each group). “n” represents the number of biologically independent experiments for each group and condition. Graphs represent mean ± s.e.m. and were compared using two-tailed Student t-test (g), one- (df, h, i) or two- (b, c) way ANOVA, followed by Tukey post hoc test (*P< 0.05, ^P< 0.01, %P< 0.005, #P< 0.0001).

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Extended Data Figure 9 CoQ10 synthesis is important for growth in 3D, but not 2D, conditions.

a, Schematic of pulse-chase study using tyrosine-derived intermediates shown in c. Cells were labelled with 13C9-Tyr for two weeks before being grown in 12C9-Tyr or 13C9-Tyr with or without unlabelled 4-HPPA, 4-HPLA, 4-HMA, and 4-HB for 24 h at 21% O2. b, Growth curve of MIAPACA2 cells with the indicated intermediates and times at 21% O2. (n= 3 technical replicates for each cell line, performed at least twice). c, Total levels and fractional labelling of unlabelled and 13C-labelled metabolites in the CoQ10 headgroup biosynthesis pathway in humans, as described in a (n=4). Endogenous 4-HB is below the limit of detection. d, Schematic of known and potential enzymes and intermediates in the CoQ10 headgroup biosynthesis pathway in humans and yeast. Dotted lines reflect potential pathways and enzymes. e, Immunoblot of the indicated MIAPACA2 cells. ERK2 is the loading control. Experiment was performed twice to check for knockout efficiency. f, Growth in 2D culture of MIAPACA2 cells (n= 4). gh, Total levels (g) and fractional labelling (h) of CoQ10 in MIAPACA2 cells. Cells were grown in 13C9-Tyr at 21% 16O2 for 24 h (n= 5). i, Growth in 3D culture of MIAPACA2 cells (n= 4) after three days. j, Representative confocal fluorescent images of the indicated MIAPACA2 cells. Images are representative of three independent experiments. k, Fractional labelling of intracellular and mitochondrial CoQ10 from 13C9-Tyr in the indicated MIAPACA2 cells (n= 4). l, Immunoblot of total, cytosolic, and mitochondrial fractions from k. Subcellular fractionation was performed twice to determine the localization of HPDL. n represents the number of biologically independent experiments for each group and condition, unless indicated (b). Graphs (mean ± s.e.m.) were compared by two-way ANOVA (b, c, fi, k), followed by Tukey (b, c, fh, k) or Dunnett’s (i) post hoc test (*P< 0.05, ^P< 0.01, %P< 0.005, #P< 0.0001).

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Extended Data Figure 10 HPDL is important for a subset of PDAC tumours.

a, Orthotopic pancreatic tumour weight from the indicated MIAPACA2 xenografts. bc, Tumour images (b) and weights (c) from a second experiment of orthotopic pancreatic tumour xenografts from MIAPACA2 cells expressing control or HPDL sgRNA with coHPDL WT or catalytically inactive mutant after 6 weeks post-injection. The first experiment set is found in a. dg, Tumour images (d, –f) and weight (eg) of orthotopic (de) or subcutaneous (fg) pancreatic tumour xenograft of PATU-8902 cells expressing control or HPDL sgRNA after 5 weeks post-injection. hi, Representative (h) and quantification (i) of H&E and immunohistochemistry for cleaved caspase 3 (CC3), phospho-histone H3 (p-HH3), and the death to proliferation ratio (CC3:p-HH3) from MIAPACA2 tumours from a. j, Overall and progression-free survival of HPDL high (n= 44) and low (n= 96) expressing PDAC tumours from the TCGA dataset. “n,” and each point represents the number of biologically independent experiments for each group and condition. Survival curve (j) was compared using the two-sided Log-rank (Mantel-Cox) test. Graphs (median ± max/min (a, c, e, g, i)) were compared by two-tailed Mann Whitney test (e, g), one-way ANOVA (a, c, i), followed by Holm-Sidak post hoc test (*P< 0.05, ^P< 0.01, %P< 0.005, #P< 0.0001).

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Extended Data Figure 11 HPDL-dependent CoQ10 biosynthesis pathway.

The canonical tyrosine catabolism, HPDL-dependent (red), and HPDL-independent (purple) CoQ10 biosynthetic pathways are shown as indicated. The HPDL-independent pathway was proposed from earlier studies in rats34. Dotted lines represent unknown pathway or transport steps. Potential intermediates and enzymes are proposed within the 4-HMA, HPDL-dependent and HPDL-independent pathways.

Extended Data Figure 12 Applications of gaseous labelling.

Our system for gaseous labelling can label cells with a wide range of isotopically labelled gases to study their incorporation into metabolites, lipids, nucleotides, proteins, and other cellular components to understand the mechanisms of the biological effects of these gases.

Supplementary information

Supplementary Information

This file contains raw western blot data for Fig. 2a–d, and Extended Data Figs. 1f, g, 7e, 9e, i.

Reporting Summary

Supplementary Table 1

List of 18O-labelled metabolites.

Supplementary Table 2

Blastp HPDL alignment descriptions.

Supplementary Table 3

Reagents.

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Banh, R.S., Kim, E.S., Spillier, Q. et al. The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway. Nature 597, 420–425 (2021). https://doi.org/10.1038/s41586-021-03865-w

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