Biology:Alpha-ketoglutarate-dependent hydroxylases

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Alpha-ketoglutarate-dependent hydroxylases are a major class of non-heme iron proteins that catalyse a wide range of reactions. These reactions include hydroxylation reactions, demethylations, ring expansions, ring closures, and desaturations.[1][2] Functionally, the αKG-dependent hydroxylases are comparable to cytochrome P450 enzymes. Both use O2 and reducing equivalents as cosubstrates and both generate water.[3]

Biological function

αKG-dependent hydroxylases have diverse roles.[4][5] In microorganisms such as bacteria, αKG-dependent dioxygenases are involved in many biosynthetic and metabolic pathways;[6][7][8] for example, in E. coli, the AlkB enzyme is associated with the repair of damaged DNA.[9][10] In plants, αKG-dependent dioxygenases are involved in diverse reactions in plant metabolism.[11] These include flavonoid biosynthesis,[12] and ethylene biosyntheses.[13] In mammals and humans, αKG-dependent dioxygenase have functional roles in biosyntheses (e.g. collagen biosynthesis[14] and L-carnitine biosynthesis[15]), post-translational modifications (e.g. protein hydroxylation[16]), epigenetic regulations (e.g. histone and DNA demethylation[17]), as well as sensors of energy metabolism.[18]

Many αKG-dependent dioxygenase also catalyse uncoupled turnover, in which oxidative decarboxylation of αKG into succinate and carbon dioxide proceeds in the absence of substrate. The catalytic activity of many αKG-dependent dioxygenases are dependent on reducing agents (especially ascorbate) although the exact roles are not understood.[19][20]

Catalytic mechanism

αKG-dependent dioxygenases catalyse oxidation reactions by incorporating a single oxygen atom from molecular oxygen (O2) into their substrates. This conversion is coupled with the oxidation of the cosubstrate αKG into succinate and carbon dioxide.[1][2] With labeled O2 as substrate, the one label appears in the succinate and one in the hydroxylated substrate:[21][22]

R3CH + O2 + −O2CC(O)CH2CH2CO2− → R3COH + CO2 + −OOCCH2CH2CO2−

The first step involves the binding of αKG and substrate to the active site. αKG coordinates as a bidentate ligand to Fe(II), while the substrate is held by noncovalent forces in close proximity. Subsequently, molecular oxygen binds end-on to Fe cis to the two donors of the αKG. The uncoordinated end of the superoxide ligand attacks the keto carbon, inducing release of CO2 and forming an Fe(IV)-oxo intermediate. This Fe=O center then oxygenates the substrate by an oxygen rebound mechanism.[1][2]

Alternative mechanisms have failed to gain support.[23]

Consensus catalytic mechanism of the αKG-dependent dioxygenase superfamily.

Structure

Protein

All αKG-dependent dioxygenases contain a conserved double-stranded β-helix (DSBH, also known as cupin) fold, which is formed with two β-sheets.[24][25]

Metallocofactor

The active site contains a highly conserved 2-His-1-carboxylate (HXD/E...H) amino acid residue triad motif, in which the catalytically-essential Fe(II) is held by two histidine residues and one aspartic acid/glutamic acid residue. The N2O triad binds to one face of the Fe center, leaving three labile sites available on the octahedron for binding αKG and O2.[1][2] A similar facial Fe-binding motif, but featuring his-his-his array, is found in cysteine dioxygenase.

Substrate and cosubstrate binding

The binding of αKG and substrate has been analyzed by X-ray crystallography, molecular dynamics calculations, and NMR spectroscopy. The binding of the ketoglutarate has been observed using enzyme inhibitors.[26]

Some αKG-dependent dioxygenases bind their substrate through an induced fit mechanism. For example, significant protein structural changes have been observed upon substrate binding for human prolyl hydroxylase isoform 2 (PHD2),[27][28][29] a αKG-dependent dioxygenase that is involved in oxygen sensing,[30] and isopenicillin N synthase (IPNS), a microbial αKG-dependent dioxygenase.[31]

Simplified view of the active site of prolyl hydroxylase isoform 2 (PHD2), a human αKG-dependent dioxygenase. The Fe(II) is coordinated by two imidazoles and one carboxylate provided by the protein. Other ligands on iron, which are transiently occupied αKG and O2, are omitted for clarity.

Inhibitors

Given the important biological roles that αKG-dependent dioxygenase play, many αKG-dependent dioxygenase inhibitors were developed. The inhibitors that were regularly used to target αKG-dependent dioxygenase include N-oxalylglycine (NOG), pyridine-2,4-dicarboxylic acid (2,4-PDCA), 5-carboxy-8-hydroxyquinoline, FG-2216 and FG-4592, which were all designed mimic the co-substrate αKG and compete against the binding of αKG at the enzyme active site Fe(II).[32][33] Although they are potent inhibitors of αKG-dependent dioxygenase, they lack selectivity and hence sometimes being referred to as so-called 'broad spectrum' inhibitors.[34] Inhibitors that compete against the substrate were also developed, such as peptidyl-based inhibitors that target human prolyl hydroxylase domain 2 (PHD2)[35] and Mildronate, a drug molecule that is commonly used in Russia and Eastern Europe that target gamma-butyrobetaine dioxygenase.[36][37][38] Finally, as αKG-dependent dioxygenases require molecular oxygen as a co-substrate, it has also been shown that gaseous molecules such as carbon monoxide[39] and nitric oxide[40][41] are inhibitors of αKG-dependent dioxygenases, presumably by competing with molecular oxygen for the binding at the active site Fe(II) ion.

Common inhibitors of αKG-dependent dioxygenases. They compete against the cosubstrate αKG for binding to the active site Fe(II).

Assays

Many assays were developed to study αKG-dependent dioxygenases so that information such as enzyme kinetics, enzyme inhibition and ligand binding can be obtained. Nuclear magnetic resonance (NMR) spectroscopy is widely applied to study αKG-dependent dioxygenases.[42] For example, assays were developed to study ligand binding,[43][44][45] enzyme kinetics,[46] modes of inhibition[47] as well as protein conformational change.[48] Mass spectrometry is also widely applied. It can be used to characterise enzyme kinetics,[49] to guide enzyme inhibitor development,[50] study ligand and metal binding[51] as well as analyse protein conformational change.[52] Assays using spectrophotometry were also used,[53] for example those that measure 2OG oxidation,[54] co-product succinate formation[55] or product formation.[56] Other biophysical techniques including (but not limited to) isothermal titration calorimetry (ITC)[57] and electron paramagnetic resonance (EPR) were also applied.[58] Radioactive assays that uses 14C labelled substrates were also developed and used.[59] Given αKG-dependent dioxygenases require oxygen for their catalytic activity, oxygen consumption assay was also applied.[60]

Further reading

References

  1. ↑ 1.0 1.1 1.2 1.3 "The most versatile of all reactive intermediates?". Nat. Chem. Biol. 3 (2): 86–87. February 2007. doi:10.1038/nchembio0207-86. PMID 17235343. 
  2. ↑ 2.0 2.1 2.2 2.3 "Fe(II)/α-ketoglutarate-dependent hydroxylases and related enzymes". Crit. Rev. Biochem. Mol. Biol. 39 (1): 21–68. January–February 2004. doi:10.1080/10409230490440541. PMID 15121720. 
  3. ↑ "Non-heme iron enzymes: contrasts to heme catalysis". Proc. Natl. Acad. Sci. U.S.A. 100 (7): 3589–3594. April 2003. doi:10.1073/pnas.0336792100. PMID 12598659. 
  4. ↑ "The iron(II) and 2-oxoacid-dependent dioxygenases and their role in metabolism". Nat. Prod. Rep. 17 (4): 367–383. August 2000. doi:10.1039/A902197C. PMID 11014338. 
  5. ↑ "Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases". Trends Biochem. Sci. 36 (1): 7–18. January 2011. doi:10.1016/j.tibs.2010.07.002. PMID 20728359. 
  6. ↑ "Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation". Proc. Natl. Acad. Sci. U.S.A. 111 (37): 13331–13336. September 2014. doi:10.1073/pnas.1409916111. PMID 25197067. Bibcode: 2014PNAS..11113331S. 
  7. ↑ "Crystal structure of carbapenem synthase (CarC)". J. Biol. Chem. 278 (23): 20843–20850. June 2003. doi:10.1074/jbc.M213054200. PMID 12611886. 
  8. ↑ "The enzymology of clavam and carbapenem biosynthesis". Chem. Commun. (34): 4251–4263. September 2005. doi:10.1039/b505964j. PMID 16113715. 
  9. ↑ Yu, Bomina; Hunt, John F. (25 August 2009). "Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB". Proceedings of the National Academy of Sciences USA 106 (34): 14315–14320. doi:10.1073/pnas.0812938106. PMID 19706517. Bibcode: 2009PNAS..10614315Y. 
  10. ↑ Ergel, Burçe; Gill, Michelle L.; Brown, Lewis; Yu, Bomina; Palmer, III, Arthur G.; Hunt, John F. (24 October 2014). "Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB". Journal of Biological Chemistry 289 (43): 29584–29601. doi:10.1074/jbc.M114.575647. PMID 25043760. 
  11. ↑ "Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism". Front. Plant Sci. 5: 524. October 2014. doi:10.3389/fpls.2014.00524. PMID 25346740. 
  12. ↑ "The function and catalysis of 2-oxoglutarate-dependent oxygenases involved in plant flavonoid biosynthesis". Int. J. Mol. Sci. 15 (1): 1080–1095. January 2014. doi:10.3390/ijms15011080. PMID 24434621. 
  13. ↑ "Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase - the ethylene-forming enzyme". Chem. Biol. 11 (10): 1383–1394. October 2004. doi:10.1016/j.chembiol.2004.08.012. PMID 15489165. 
  14. ↑ "Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis". Matrix Biol. 22 (1): 15–24. March 2003. doi:10.1016/S0945-053X(03)00006-4. PMID 12714038. 
  15. ↑ "Structural and mechanistic studies on γ-butyrobetaine hydroxylase". Chem. Biol. 17 (12): 1316–1324. December 2010. doi:10.1016/j.chembiol.2010.09.016. PMID 21168767. 
  16. ↑ Markolovic, Suzana; Wilkins, Sarah E.; Schofield, Christopher J. (2015-08-21). "Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases". The Journal of Biological Chemistry 290 (34): 20712–20722. doi:10.1074/jbc.R115.662627. ISSN 1083-351X. PMID 26152730. 
  17. ↑ "Mechanisms of human histone and nucleic acid demethylases". Curr. Opin. Chem. Biol. 16 (5–6): 525–534. December 2012. doi:10.1016/j.cbpa.2012.09.015. PMID 23063108. 
  18. ↑ Salminen, A; Kauppinen, A; Kaarniranta, K (2015). "2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism, oxygen availability, and iron homeostasis: potential role in the regulation of aging process". Cell Mol Life Sci 72 (20): 3897–914. doi:10.1007/s00018-015-1978-z. PMID 26118662. 
  19. ↑ "Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase". J. Biol. Chem. 259 (9): 5403–5405. May 1984. doi:10.1016/S0021-9258(18)91023-9. PMID 6325436. 
  20. ↑ "Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents". Biochem. J. 427 (1): 135–142. March 2010. doi:10.1042/BJ20091609. PMID 20055761. https://hal.archives-ouvertes.fr/hal-00479275/file/PEER_stage2_10.1042%252FBJ20091609.pdf. 
  21. ↑ "Incorporation of oxygen into the succinate co-product of iron(II) and 2-oxoglutarate dependent oxygenases from bacteria, plants and humans". FEBS Lett. 579 (23): 5170–5174. September 2005. doi:10.1016/j.febslet.2005.08.033. PMID 16153644. 
  22. ↑ "Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV=HO species". Proc. Natl. Acad. Sci. U.S.A. 107 (9): 3982–3987. March 2010. doi:10.1073/pnas.0911565107. PMID 20147623. 
  23. ↑ "Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases". Biochemistry 53 (15): 2483–2493. April 2014. doi:10.1021/bi500086p. PMID 24684493. 
  24. ↑ "Structural studies on human 2-oxoglutarate dependent oxygenases". Curr. Opin. Struct. Biol. 20 (6): 659–672. December 2010. doi:10.1016/j.sbi.2010.08.006. PMID 20888218. 
  25. ↑ "Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins". J. Inorg. Biochem. 100 (4): 644–669. April 2006. doi:10.1016/j.jinorgbio.2006.01.024. PMID 16513174. 
  26. ↑ You, Z.; Omura, S.; Ikeda, H.; Cane, D.E.; Jogl, G. (2007). "Crystal structure of the non-heme iron dioxygenase PtlH in pentalenolactone biosynthesis". J. Biol. Chem. 282 (2): 36552–60. doi:10.1074/jbc.M706358200. PMID 17942405. 
  27. ↑ "Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2)". Proc. Natl. Acad. Sci. U.S.A. 103 (26): 9814–9819. June 2006. doi:10.1073/pnas.0601283103. PMID 16782814. Bibcode: 2006PNAS..103.9814M. 
  28. ↑ "Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases". Structure 17 (7): 981–989. July 2009. doi:10.1016/j.str.2009.06.002. PMID 19604478. 
  29. ↑ "Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases". Nat. Commun. 7: 12673. August 2016. doi:10.1038/ncomms12673. PMID 27561929. Bibcode: 2016NatCo...712673C. 
  30. ↑ "The prolyl hydroxylase enzymes that act as oxygen sensors regulating destruction of hypoxia-inducible factor α". Advan. Enzyme Regul. 44: 75–92. 2004. doi:10.1016/j.advenzreg.2003.11.017. PMID 15581484. 
  31. ↑ "Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation". Nature 387 (6635): 827–830. June 1997. doi:10.1038/42990. PMID 9194566. 
  32. ↑ "Inhibition of 2-oxoglutarate dependent oxygenases". Chem. Soc. Rev. 40 (8): 4364–4397. August 2011. doi:10.1039/c0cs00203h. PMID 21390379. 
  33. ↑ "Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials". Chem. Sci. 8 (11): 7651–7668. September 2017. doi:10.1039/C7SC02103H. PMID 29435217. 
  34. ↑ "5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation". Chem. Sci. 4 (8): 3110–3117. August 2013. doi:10.1039/C3SC51122G. PMID 26682036. 
  35. ↑ "Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides". Bioorg. Med. Chem. Lett. 21 (14): 4325–4328. July 2011. doi:10.1016/j.bmcl.2011.05.050. PMID 21665470. 
  36. ↑ "Mildronate, a novel fatty acid oxidation inhibitor and antianginal agent, reduces myocardial infarct size without affecting hemodynamics". Journal of Cardiovascular Pharmacology 47 (3): 493–9. Mar 2006. doi:10.1097/01.fjc.0000211732.76668.d2. PMID 16633095. 
  37. ↑ "Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction". Journal of Cardiovascular Pharmacology 48 (6): 314–9. Dec 2006. doi:10.1097/01.fjc.0000250077.07702.23. PMID 17204911. 
  38. ↑ "Beneficial effects of MET-88, a gamma-butyrobetaine hydroxylase inhibitor in rats with heart failure following myocardial infarction". European Journal of Pharmacology 395 (3): 217–24. May 2000. doi:10.1016/S0014-2999(00)00098-4. PMID 10812052. 
  39. ↑ "Carbon monoxide is an inhibitor of HIF prolyl hydroxylase domain 2". ChemBioChem 22 (15): 2521–2525. June 2021. doi:10.1002/cbic.202100181. PMID 34137488. 
  40. ↑ "Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases". Molecular Biology of the Cell 14 (8): 3470–3481. August 2003. doi:10.1091/mbc.E02-12-0791. PMID 12925778. 
  41. ↑ "Nitric oxide modulates oxygen sensing by hypoxia-inducible factor 1-dependent induction of prolyl hydroxylase 2". Journal of Biological Chemistry 282 (3): 1788–1796. January 2007. doi:10.1074/jbc.M607065200. PMID 17060326. 
  42. ↑ "NMR studies of the non-haem Fe(II) and 2-oxoglutarate-dependent oxygenases". J. Inorg. Biochem. 177: 384–394. December 2017. doi:10.1016/j.jinorgbio.2017.08.032. PMID 28893416. 
  43. ↑ "Reporter ligand NMR screening method for 2-oxoglutarate oxygenase inhibitors". J. Med. Chem. 56 (2): 547–555. January 2013. doi:10.1021/jm301583m. PMID 23234607. 
  44. ↑ "Using NMR solvent water relaxation to investigate metalloenzyme-ligand binding interactions". J. Med. Chem. 53 (2): 867–875. January 2010. doi:10.1021/jm901537q. PMID 20025281. 
  45. ↑ "Development and application of ligand-based NMR screening assays for γ-butyrobetaine hydroxylase". Med. Chem. Commun. 7 (5): 873–880. February 2017. doi:10.1039/C6MD00004E. 
  46. ↑ "Monitoring the activity of 2-oxoglutarate dependent histone demethylases by NMR spectroscopy: direct observation of formaldehyde". ChemBioChem 11 (4): 506–510. March 2010. doi:10.1002/cbic.200900713. PMID 20095001. 
  47. ↑ "Different modes of inhibitor binding to prolyl hydroxylase by combined use of X-ray crystallography and NMR spectroscopy of paramagnetic complexes". J. Am. Chem. Soc. 131 (46): 16654–16655. November 2009. doi:10.1021/ja907933p. PMID 19886658. https://figshare.com/articles/journal_contribution/2810647. 
  48. ↑ "Dynamic states of the DNA repair enzyme AlkB regulate product release". EMBO Rep. 9 (9): 872–877. September 2008. doi:10.1038/embor.2008.120. PMID 18617893. 
  49. ↑ "Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases". J. Biol. Chem. 283 (7): 3808–3815. February 2008. doi:10.1074/jbc.M707411200. PMID 18063574. 
  50. ↑ "Dynamic combinatorial chemistry employing boronic acids/boronate esters leads to potent oxygenase inhibitors". Angew. Chem. Int. Ed. 51 (27): 6672–6675. July 2012. doi:10.1002/anie.201202000. PMID 22639232. 
  51. ↑ "ESI-MS studies on prolyl hydroxylase domain 2 reveal a new metal binding site". ChemMedChem 3 (4): 569–572. April 2008. doi:10.1002/cmdc.200700233. PMID 18058781. 
  52. ↑ "Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure". J. Med. Chem. 52 (9): 2799–2805. May 2009. doi:10.1021/jm900285r. PMID 19364117. 
  53. ↑ "Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study". J. Biol. Inorg. Chem. 22 (2–3): 367–379. April 2016. doi:10.1007/s00775-016-1406-3. PMID 27812832. 
  54. ↑ "A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases". Anal. Biochem. 336 (1): 125–131. January 2005. doi:10.1016/j.ab.2004.09.019. PMID 15582567. 
  55. ↑ "An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation". Anal. Biochem. 353 (1): 69–74. June 2006. doi:10.1016/j.ab.2006.03.033. PMID 16643838. 
  56. ↑ "Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase". ChemBioChem 13 (11): 1559–1563. July 2012. doi:10.1002/cbic.201200256. PMID 22730246. 
  57. ↑ "The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase". Biochem. J. 473 (9): 1179–1189. May 2016. doi:10.1042/BCJ20160146. PMID 26936969. http://opus.bath.ac.uk/49739/1/Supp_Fig_all.pdf. 
  58. ↑ "Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH)". J. Inorg. Biochem. 113: 25–30. August 2012. doi:10.1016/j.jinorgbio.2012.03.002. PMID 22687491. 
  59. ↑ "Assay of prolyl 4-hydroxylase by the chromatographic determination of [14Csuccinic acid on ion-exchange minicolumns"]. Biochem. J. 240 (2): 617–619. December 1986. doi:10.1042/bj2400617. PMID 3028379. 
  60. ↑ "Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay". Biochem. J. 401 (1): 227–234. January 2007. doi:10.1042/BJ20061151. PMID 16952279. 



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