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'''Alpha-ketoglutarate-dependent hydroxylases''' are non-heme, iron-containing [[enzyme]]s that consume oxygen and [[alpha-ketoglutarate]] (αKG, also known as 2-oxoglutarate, or 2OG) as co-substrates. They catalyse a wide range of oxygenation reactions. These include hydroxylation reactions, demethylations, ring expansions, ring closures and desaturations.<ref name="pmidunknown01">{{cite journal |vauthors=Flashman E, Schofield CJ | title = The most versatile of all reactive intermediates? | journal = Nat. Chem. Biol. | volume = 3 | issue = 2| pages = 86–87 |date=February 2007 | pmid = 17235343| doi = 10.1038/nchembio0207-86 }}</ref><ref name="pmid15121720">{{cite journal |vauthors=Hausinger RP | title = Fe(II)/α-ketoglutarate-dependent hydroxylases and related enzymes | journal = Crit. Rev. Biochem. Mol. Biol. | volume = 39 | issue = 1 | pages = 21–68 |date=January–February 2004 | pmid =15121720 | doi = 10.1080/10409230490440541 | s2cid = 85784668 }}</ref> Functionally, the αKG-dependent hydroxylases are comparable to [[cytochrome P450]] enzymes, which use oxygen and reducing equivalents to oxygenate substrates concomitant with formation of water.<ref name="pmid12598659">{{cite journal |vauthors= Solomon EI, Decker A, Lehnert N | title = Non-heme iron enzymes: contrasts to heme catalysis | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 100 | issue = 7 | pages = 3589–3594 |date=April 2003 | pmid = 12598659 | doi = 10.1073/pnas.0336792100 | pmc=152966}}</ref>
'''Alpha-ketoglutarate-dependent hydroxylases''' are a major class of [[non-heme iron protein]]s that catalyse a wide range of reactions. These reactions include hydroxylation reactions, demethylations, ring expansions, ring closures, and desaturations.<ref name="pmidunknown01">{{cite journal |vauthors=Flashman E, [[Christopher J. Schofield|Schofield CJ]] | title = The most versatile of all reactive intermediates? | journal = Nat. Chem. Biol. | volume = 3 | issue = 2| pages = 86–87 |date=February 2007 | pmid = 17235343| doi = 10.1038/nchembio0207-86 | doi-access = free }}</ref><ref name="pmid15121720">{{cite journal |vauthors=Hausinger RP | title = Fe(II)/α-ketoglutarate-dependent hydroxylases and related enzymes | journal = Crit. Rev. Biochem. Mol. Biol. | volume = 39 | issue = 1 | pages = 21–68 |date=January–February 2004 | pmid =15121720 | doi = 10.1080/10409230490440541 | s2cid = 85784668 }}</ref> Functionally, the αKG-dependent hydroxylases are comparable to [[cytochrome P450]] enzymes. Both use O<sub>2</sub> and reducing equivalents as cosubstrates and both generate water.<ref name="pmid12598659">{{cite journal |vauthors= Solomon EI, Decker A, Lehnert N | title = Non-heme iron enzymes: contrasts to heme catalysis | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 100 | issue = 7 | pages = 3589–3594 |date=April 2003 | pmid = 12598659 | doi = 10.1073/pnas.0336792100 | pmc=152966| doi-access = free }}</ref>


==Biological function==
==Biological function==
αKG-dependent hydroxylases have diverse roles.<ref name="pmid11014338">{{cite journal |vauthors=Prescott AG, Lloyd MD | title = The iron(II) and 2-oxoacid-dependent dioxygenases and their role in metabolism | journal = Nat. Prod. Rep. | volume = 17 | issue = 4 | pages = 367–383 |date=August 2000 | pmid = 11014338 | doi = 10.1039/A902197C }}</ref><ref name="pmid20728359">{{cite journal |vauthors=Loenarz C, Schofield CJ | title = Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases | journal = Trends Biochem. Sci. | volume = 36 | issue = 1 | pages = 7–18 |date=January 2011 | pmid = 20728359 | doi = 10.1016/j.tibs.2010.07.002 }}</ref> In microorganisms such as bacteria, αKG-dependent dioxygenases are involved in many biosynthetic pathways.<ref name="pmid25197067">{{cite journal |vauthors=Scotti JS, Leung IK, Ge W, Bentley MA, Paps J, Kramer HB, Lee J, Aik W, Choi H, Paulsen SM, Bowman LA, Loik ND, Horita S, Ho CH, Kershaw NJ, Tang CM, Claridge TD, Preston GM, McDonough MA, Schofield CJ | title = Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 111 | issue = 37 | pages = 13331–13336 |date=September 2014 | pmid = 25197067 | doi = 10.1073/pnas.1409916111 | pmc=4169948| bibcode = 2014PNAS..11113331S }}</ref><ref name="pmid12611886">{{cite journal |vauthors=Clifton IJ, Doan LX, Sleeman MC, Topf M, Suzuki H, Wilmouth RC, Schofield CJ | title = Crystal structure of carbapenem synthase (CarC) | journal = J. Biol. Chem. | volume = 278 | issue = 23 | pages = 20843–20850 |date=June 2003 | pmid = 12611886 | doi = 10.1074/jbc.M213054200 | s2cid = 9662423 }}</ref><ref name="pmid16113715">{{cite journal |vauthors=Kershaw NJ, Caines ME, Sleeman MC, Schofield CJ | title = The enzymology of clavam and carbapenem biosynthesis | journal = Chem. Commun. | volume = | issue = 34| pages = 4251–4263 |date=September 2005 | pmid = 16113715 | doi = 10.1039/b505964j }}</ref> In plants, αKG-dependent dioxygenases are involved in many different reactions in plant metabolism.<ref name="pmid25346740">{{cite journal |vauthors=Farrow SC, Facchini PJ | title = Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism | journal = Front. Plant Sci. | volume = 5 | issue = | pages = 524 |date=October 2014 | pmid = 25346740 | doi = 10.3389/fpls.2014.00524 | pmc=4191161}}</ref> These include flavonoid biosynthesis,<ref name="pmid24434621">{{cite journal |vauthors=Cheng AX, Han XJ, Wu YF, Lou HX | title = The function and catalysis of 2-oxoglutarate-dependent oxygenases involved in plant flavonoid biosynthesis | journal = Int. J. Mol. Sci. | volume = 15 | issue = 1 | pages = 1080–1095 |date=January 2014 | pmid = 24434621 | doi = 10.3390/ijms15011080 | pmc=3907857}}</ref> and ethylene biosyntheses.<ref name="pmid15489165">{{cite journal |vauthors=Zhang Z, Ren JS, Clifton IJ, Schofield CJ | title = Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase - the ethylene-forming enzyme | journal = Chem. Biol. | volume = 11 | issue = 10 | pages = 1383–1394 |date=October 2004 | pmid = 15489165 | doi = 10.1016/j.chembiol.2004.08.012 }}</ref> In mammals and humans, αKG-dependent dioxygenase have functional roles in biosyntheses (e.g. collagen biosynthesis<ref name="pmid12714038">{{cite journal |vauthors=Myllyharju J | title = Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis | journal = Matrix Biol. | volume = 22 | issue = 1 | pages = 15–24 |date=March 2003 | pmid = 12714038 | doi = 10.1016/S0945-053X(03)00006-4 }}</ref> and L-carnitine biosynthesis<ref name="pmid21168767 ">{{cite journal |vauthors=Leung IK, Krojer TJ, Kochan GT, Henry L, von Delft F, Claridge TD, Oppermann U, McDonough MA, Schofield CJ | title = Structural and mechanistic studies on γ-butyrobetaine hydroxylase | journal = Chem. Biol. | volume = 17 | issue = 12 | pages = 1316–1324 |date=December 2010 | pmid = 21168767 | doi = 10.1016/j.chembiol.2010.09.016 }}</ref>), post-translational modifications (e.g. protein hydroxylation<ref>{{Cite journal|last1=Markolovic|first1=Suzana|last2=Wilkins|first2=Sarah E.|last3=Schofield|first3=Christopher J.|date=2015-08-21|title=Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases|journal=The Journal of Biological Chemistry|volume=290|issue=34|pages=20712–20722|doi=10.1074/jbc.R115.662627|issn=1083-351X|pmc=4543633|pmid=26152730}}</ref>), epigenetic regulations (e.g. [[histone]] and [[DNA]] demethylation<ref name="pmid23063108 ">{{cite journal |vauthors=Walport LJ, Hopkinson RJ, Schofield CJ | title = Mechanisms of human histone and nucleic acid demethylases | journal = Curr. Opin. Chem. Biol. | volume = 16 | issue = 5–6 | pages = 525–534 |date=December 2012 | pmid = 23063108 | doi = 10.1016/j.cbpa.2012.09.015 }}</ref>), as well as sensors of [[cellular respiration|energy metabolism]].<ref name="MyUser_Ncbi.nlm.nih.gov_July_24_2015c">{{cite journal |title= 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism, oxygen availability, and iron homeostasis: potential role in the regulation of aging process| pmid=26118662 | doi=10.1007/s00018-015-1978-z | volume=72 | issue=20 | journal=Cell Mol Life Sci | pages=3897–914 | last1 = Salminen | first1 = A | last2 = Kauppinen | first2 = A | last3 = Kaarniranta | first3 = K | year=2015| s2cid=14310267 }}</ref>
αKG-dependent hydroxylases have diverse roles.<ref name="pmid11014338">{{cite journal |vauthors=Prescott AG, Lloyd MD | title = The iron(II) and 2-oxoacid-dependent dioxygenases and their role in metabolism | journal = Nat. Prod. Rep. | volume = 17 | issue = 4 | pages = 367–383 |date=August 2000 | pmid = 11014338 | doi = 10.1039/A902197C }}</ref><ref name="pmid20728359">{{cite journal |vauthors=Loenarz C, [[Christopher J. Schofield|Schofield CJ]] | title = Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases | journal = Trends Biochem. Sci. | volume = 36 | issue = 1 | pages = 7–18 |date=January 2011 | pmid = 20728359 | doi = 10.1016/j.tibs.2010.07.002 }}</ref> In microorganisms such as bacteria, αKG-dependent dioxygenases are involved in many biosynthetic and metabolic pathways;<ref name="pmid25197067">{{cite journal |vauthors=Scotti JS, Leung IK, Ge W, Bentley MA, Paps J, Kramer HB, Lee J, Aik W, Choi H, Paulsen SM, Bowman LA, Loik ND, Horita S, Ho CH, Kershaw NJ, Tang CM, Claridge TD, Preston GM, McDonough MA, [[Christopher J. Schofield|Schofield CJ]] | title = Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 111 | issue = 37 | pages = 13331–13336 |date=September 2014 | pmid = 25197067 | doi = 10.1073/pnas.1409916111 | pmc=4169948| bibcode = 2014PNAS..11113331S | doi-access = free }}</ref><ref name="pmid12611886">{{cite journal |vauthors=Clifton IJ, Doan LX, Sleeman MC, Topf M, Suzuki H, Wilmouth RC, [[Christopher J. Schofield|Schofield CJ]] | title = Crystal structure of carbapenem synthase (CarC) | journal = J. Biol. Chem. | volume = 278 | issue = 23 | pages = 20843–20850 |date=June 2003 | pmid = 12611886 | doi = 10.1074/jbc.M213054200 | s2cid = 9662423 | doi-access = free }}</ref><ref name="pmid16113715">{{cite journal |vauthors=Kershaw NJ, Caines ME, Sleeman MC, [[Christopher J. Schofield|Schofield CJ]] | title = The enzymology of clavam and carbapenem biosynthesis | journal = Chem. Commun. | issue = 34| pages = 4251–4263 |date=September 2005 | pmid = 16113715 | doi = 10.1039/b505964j }}</ref> for example, in [[Escherichia coli |''E. coli'']], the [[AlkB]] enzyme is associated with the repair of damaged [[DNA]].<ref name=Yu >{{cite journal |last1=Yu |first1=Bomina |last2=Hunt |first2=John F. |date=25 August 2009 |title=Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB |journal=Proceedings of the National Academy of Sciences USA |volume=106 |issue=34 |pages=14315–14320 |doi=10.1073/pnas.0812938106 |pmid=19706517 |pmc=2725012 |bibcode=2009PNAS..10614315Y |doi-access=free }}</ref><ref name=Ergel >{{cite journal |last1=Ergel |first1=Burçe |last2=Gill |first2=Michelle L. |last3=Brown |first3=Lewis |last4=Yu |first4=Bomina |last5=Palmer, III |first5=Arthur G. |last6=Hunt |first6=John F. |date=24 October 2014 |title=Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB |journal=Journal of Biological Chemistry |volume=289 |issue=43 |pages=29584–29601 |doi=10.1074/jbc.M114.575647 |pmid=25043760 |pmc=4207975 |doi-access=free }}</ref> In plants, αKG-dependent dioxygenases are involved in diverse reactions in plant metabolism.<ref name="pmid25346740">{{cite journal |vauthors=Farrow SC, Facchini PJ | title = Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism | journal = Front. Plant Sci. | volume = 5 | pages = 524 |date=October 2014 | pmid = 25346740 | doi = 10.3389/fpls.2014.00524 | pmc=4191161| doi-access = free }}</ref> These include flavonoid biosynthesis,<ref name="pmid24434621">{{cite journal |vauthors=Cheng AX, Han XJ, Wu YF, Lou HX | title = The function and catalysis of 2-oxoglutarate-dependent oxygenases involved in plant flavonoid biosynthesis | journal = Int. J. Mol. Sci. | volume = 15 | issue = 1 | pages = 1080–1095 |date=January 2014 | pmid = 24434621 | doi = 10.3390/ijms15011080 | pmc=3907857| doi-access = free }}</ref> and ethylene biosyntheses.<ref name="pmid15489165">{{cite journal |vauthors=Zhang Z, Ren JS, Clifton IJ, [[Christopher J. Schofield|Schofield CJ]] | title = Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase - the ethylene-forming enzyme | journal = Chem. Biol. | volume = 11 | issue = 10 | pages = 1383–1394 |date=October 2004 | pmid = 15489165 | doi = 10.1016/j.chembiol.2004.08.012 | doi-access = free }}</ref> In mammals and humans, αKG-dependent dioxygenase have functional roles in biosyntheses (e.g. collagen biosynthesis<ref name="pmid12714038">{{cite journal |vauthors=Myllyharju J | title = Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis | journal = Matrix Biol. | volume = 22 | issue = 1 | pages = 15–24 |date=March 2003 | pmid = 12714038 | doi = 10.1016/S0945-053X(03)00006-4 }}</ref> and L-carnitine biosynthesis<ref name="pmid21168767 ">{{cite journal |vauthors=Leung IK, Krojer TJ, Kochan GT, Henry L, von Delft F, Claridge TD, Oppermann U, McDonough MA, [[Christopher J. Schofield|Schofield CJ]] | title = Structural and mechanistic studies on γ-butyrobetaine hydroxylase | journal = Chem. Biol. | volume = 17 | issue = 12 | pages = 1316–1324 |date=December 2010 | pmid = 21168767 | doi = 10.1016/j.chembiol.2010.09.016 | doi-access = free }}</ref>), post-translational modifications (e.g. protein hydroxylation<ref>{{Cite journal|last1=Markolovic|first1=Suzana|last2=Wilkins|first2=Sarah E.|last3=Schofield|first3=Christopher J.|author3-link=Christopher J. Schofield|date=2015-08-21|title=Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases|journal=The Journal of Biological Chemistry|volume=290|issue=34|pages=20712–20722|doi=10.1074/jbc.R115.662627|issn=1083-351X|pmc=4543633|pmid=26152730|doi-access=free}}</ref>), epigenetic regulations (e.g. [[histone]] and [[DNA]] demethylation<ref name="pmid23063108 ">{{cite journal |vauthors=Walport LJ, Hopkinson RJ, [[Christopher J. Schofield|Schofield CJ]] | title = Mechanisms of human histone and nucleic acid demethylases | journal = Curr. Opin. Chem. Biol. | volume = 16 | issue = 5–6 | pages = 525–534 |date=December 2012 | pmid = 23063108 | doi = 10.1016/j.cbpa.2012.09.015 }}</ref>), as well as sensors of [[cellular respiration|energy metabolism]].<ref name="MyUser_Ncbi.nlm.nih.gov_July_24_2015c">{{cite journal |title= 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism, oxygen availability, and iron homeostasis: potential role in the regulation of aging process| pmid=26118662 | doi=10.1007/s00018-015-1978-z | volume=72 | issue=20 | journal=Cell Mol Life Sci | pages=3897–914 | last1 = Salminen | first1 = A | last2 = Kauppinen | first2 = A | last3 = Kaarniranta | first3 = K | year=2015| s2cid=14310267 | pmc=11114064 }}</ref>


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.<ref name="pmid6325436 ">{{cite journal |vauthors=Myllylä R, Majamaa K, Günzler V, Hanauske-Abel HM, Kivirikko KI | title = Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase | journal = J. Biol. Chem. | volume = 259 | issue = 9 | pages = 5403–5405 |date=May 1984 | pmid = 6325436 | doi = }}</ref><ref name="pmid20055761 ">{{cite journal |vauthors= Flashman E, Davies SL, Yeoh KK, Schofield CJ | title = Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents | journal = Biochem. J. | volume = 427 | issue = 1 | pages = 135–142 |date=March 2010 | pmid = 20055761 | doi = 10.1042/BJ20091609 | url = https://hal.archives-ouvertes.fr/hal-00479275/file/PEER_stage2_10.1042%252FBJ20091609.pdf }}</ref>
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.<ref name="pmid6325436 ">{{cite journal |vauthors=Myllylä R, Majamaa K, Günzler V, Hanauske-Abel HM, Kivirikko KI | title = Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase | journal = J. Biol. Chem. | volume = 259 | issue = 9 | pages = 5403–5405 |date=May 1984 | doi = 10.1016/S0021-9258(18)91023-9 | pmid = 6325436 | doi-access = free }}</ref><ref name="pmid20055761 ">{{cite journal |vauthors= Flashman E, Davies SL, Yeoh KK, [[Christopher J. Schofield|Schofield CJ]] | title = Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents | journal = Biochem. J. | volume = 427 | issue = 1 | pages = 135–142 |date=March 2010 | pmid = 20055761 | doi = 10.1042/BJ20091609 | url = https://hal.archives-ouvertes.fr/hal-00479275/file/PEER_stage2_10.1042%252FBJ20091609.pdf }}</ref>


==Catalytic mechanism==
==Catalytic mechanism==
αKG-dependent dioxygenases catalyse oxidation reactions by incorporating a single oxygen atom from molecular oxygen (O<sub>2</sub>) into their substrates. This conversion is coupled with the oxidation of the cosubstrate [[2-oxoglutarate|αKG]] into succinate and carbon dioxide.<ref name="pmidunknown01"/><ref name="pmid15121720"/> With labeled O<sub>2</sub> as substrate, the one label appears in the succinate and one in the hydroxylated substrate:<ref name="pmid16153644">{{cite journal |vauthors= Welford RW, Kirkpatrick JM, McNeill LA, Puri M, Oldham NJ, Schofield CJ | title = Incorporation of oxygen into the succinate co-product of iron(II) and 2-oxoglutarate dependent oxygenases from bacteria, plants and humans | journal = FEBS Lett. | volume = 579 | issue = 23 | pages = 5170–5174 |date=September 2005 | pmid = 16153644 | doi = 10.1016/j.febslet.2005.08.033 | hdl = 10536/DRO/DU:30019701 | s2cid = 11295236 }}</ref><ref name="pmid20147623">{{cite journal |vauthors=Grzyska PK, Appelman EH, Hausinger RP, Proshlyakov DA | title = Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV=HO species | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 107 | issue = 9 | pages = 3982–3987 |date=March 2010 | pmid = 20147623 | doi = 10.1073/pnas.0911565107 | pmc=2840172}}</ref><ref>{{Cite journal|last=Menon|first=Binuraj R. K.|last2=Richmond|first2=Daniel|last3=Menon|first3=Navya|date=2020-10-15|title=Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals|url=https://www.tandfonline.com/doi/full/10.1080/01614940.2020.1823788|journal=Catalysis Reviews|language=en|pages=1–59|doi=10.1080/01614940.2020.1823788|issn=0161-4940}}</ref>
αKG-dependent dioxygenases catalyse oxidation reactions by incorporating a single oxygen atom from molecular oxygen (O<sub>2</sub>) into their substrates. This conversion is coupled with the oxidation of the cosubstrate [[2-oxoglutarate|αKG]] into succinate and carbon dioxide.<ref name="pmidunknown01"/><ref name="pmid15121720"/> With labeled O<sub>2</sub> as substrate, the one label appears in the succinate and one in the hydroxylated substrate:<ref name="pmid16153644">{{cite journal |vauthors= Welford RW, Kirkpatrick JM, McNeill LA, Puri M, Oldham NJ, [[Christopher J. Schofield|Schofield CJ]] | title = Incorporation of oxygen into the succinate co-product of iron(II) and 2-oxoglutarate dependent oxygenases from bacteria, plants and humans | journal = FEBS Lett. | volume = 579 | issue = 23 | pages = 5170–5174 |date=September 2005 | pmid = 16153644 | doi = 10.1016/j.febslet.2005.08.033 | hdl = 10536/DRO/DU:30019701 | s2cid = 11295236 | hdl-access = free }}</ref><ref name="pmid20147623">{{cite journal |vauthors=Grzyska PK, Appelman EH, Hausinger RP, Proshlyakov DA | title = Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV=HO species | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 107 | issue = 9 | pages = 3982–3987 |date=March 2010 | pmid = 20147623 | doi = 10.1073/pnas.0911565107 | pmc=2840172|doi-access = free}}</ref>
:R<sub>3</sub>CH + <span style="color:red;">O<sub>2</sub></span> + <sup>−</sup>O<sub>2</sub>CC(O)CH<sub>2</sub>CH<sub>2</sub>CO<sub>2</sub><sup>−</sup> → R<sub>3</sub>C<span style="color:red;">O</span>H + CO<sub>2</sub> + <sup>−</sup>O<span style="color:red;">O</span>CCH<sub>2</sub>CH<sub>2</sub>CO<sub>2</sub><sup>−</sup>
:R<sub>3</sub>CH + <span style="color:red;">O<sub>2</sub></span> + <sup>−</sup>O<sub>2</sub>CC(O)CH<sub>2</sub>CH<sub>2</sub>CO<sub>2</sub><sup>−</sup> → R<sub>3</sub>C<span style="color:red;">O</span>H + CO<sub>2</sub> + <sup>−</sup>O<span style="color:red;">O</span>CCH<sub>2</sub>CH<sub>2</sub>CO<sub>2</sub><sup>−</sup>


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 CO<sub>2</sub> and forming an [[metal oxo complex|Fe(IV)-oxo intermediate]]. This Fe=O center then oxygenates the substrate by an [[oxygen rebound mechanism]].<ref name="pmidunknown01"/><ref name="pmid15121720"/>
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 CO<sub>2</sub> and forming an [[metal oxo complex|Fe(IV)-oxo intermediate]]. This Fe=O center then oxygenates the substrate by an [[oxygen rebound mechanism]].<ref name="pmidunknown01"/><ref name="pmid15121720"/>


Alternative mechanisms have failed to gain support.<ref name="pmid24684493">{{cite journal |vauthors= Tarhonskaya H, Szöllössi A, Leung IK, Bush JT, Henry L, Chowdhury R, Iqbal A, Claridge TD, Schofield CJ, Flashman E | title = Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases | journal = Biochemistry | volume = 53 | issue = 15 | pages = 2483–2493 |date=April 2014 | pmid = 24684493 | doi = 10.1021/bi500086p }}</ref>
Alternative mechanisms have failed to gain support.<ref name="pmid24684493">{{cite journal |vauthors= Tarhonskaya H, Szöllössi A, Leung IK, Bush JT, Henry L, Chowdhury R, Iqbal A, Claridge TD, [[Christopher J. Schofield|Schofield CJ]], Flashman E | title = Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases | journal = Biochemistry | volume = 53 | issue = 15 | pages = 2483–2493 |date=April 2014 | pmid = 24684493 | doi = 10.1021/bi500086p }}</ref>


[[File:Mechanism of 2OG oxygenases.png|thumb|center|upright=5|Consensus catalytic mechanism of the αKG-dependent dioxygenase superfamily.]]
[[File:Mechanism of 2OG oxygenases.png|thumb|center|upright=5|Consensus catalytic mechanism of the αKG-dependent dioxygenase superfamily.]]
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==Structure==
==Structure==
===Protein===
===Protein===
All αKG-dependent dioxygenases contain a conserved [[Jelly roll fold|double-stranded β-helix]] (DSBH, also known as cupin) fold, which is formed with two β-sheets.<ref name="pmid20888218">{{cite journal |vauthors= McDonough MA, Loenarz C, Chowdhury R, Clifton IJ, Schofield CJ | title = Structural studies on human 2-oxoglutarate dependent oxygenases | journal = Curr. Opin. Struct. Biol. | volume = 20 | issue = 6 | pages = 659–672 |date=December 2010 | pmid = 20888218 | doi = 10.1016/j.sbi.2010.08.006 }}</ref><ref name="pmid16513174">{{cite journal |vauthors= Clifton IJ, McDonough MA, Ehrismann D, Kershaw NJ, Granatino N, Schofield CJ | title = Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins | journal = J. Inorg. Biochem. | volume = 100 | issue = 4 | pages = 644–669 |date=April 2006 | pmid = 16513174 | doi = 10.1016/j.jinorgbio.2006.01.024 }}</ref>
All αKG-dependent dioxygenases contain a conserved [[double-stranded β-helix]] (DSBH, also known as cupin) fold, which is formed with two β-sheets.<ref name="pmid20888218">{{cite journal |vauthors= McDonough MA, Loenarz C, Chowdhury R, Clifton IJ, [[Christopher J. Schofield|Schofield CJ]] | title = Structural studies on human 2-oxoglutarate dependent oxygenases | journal = Curr. Opin. Struct. Biol. | volume = 20 | issue = 6 | pages = 659–672 |date=December 2010 | pmid = 20888218 | doi = 10.1016/j.sbi.2010.08.006 }}</ref><ref name="pmid16513174">{{cite journal |vauthors= Clifton IJ, McDonough MA, Ehrismann D, Kershaw NJ, Granatino N, [[Christopher J. Schofield|Schofield CJ]] | title = Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins | journal = J. Inorg. Biochem. | volume = 100 | issue = 4 | pages = 644–669 |date=April 2006 | pmid = 16513174 | doi = 10.1016/j.jinorgbio.2006.01.024 }}</ref>


===Metallocofactor===
===Metallocofactor===
Line 25: Line 25:
===Substrate and cosubstrate binding===
===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.<ref>{{cite journal|journal=J. Biol. Chem.|year=2007|volume=282|issue=2|pages=36552–60|title=Crystal structure of the non-heme iron dioxygenase PtlH in pentalenolactone biosynthesis|author1=You, Z. |author2=Omura, S. |author3=Ikeda, H. |author4=Cane, D.E. |author5=Jogl, G.|doi=10.1074/jbc.M706358200|pmid=17942405|pmc=3010413}}</ref>
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.<ref>{{cite journal|journal=J. Biol. Chem.|year=2007|volume=282|issue=2|pages=36552–60|title=Crystal structure of the non-heme iron dioxygenase PtlH in pentalenolactone biosynthesis|author1=You, Z. |author2=Omura, S. |author3=Ikeda, H. |author4=Cane, D.E. |author5=Jogl, G.|doi=10.1074/jbc.M706358200|pmid=17942405|pmc=3010413|doi-access=free}}</ref>


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 [[EGLN1|prolyl hydroxylase isoform 2]] (PHD2),<ref name="pmid16782814">{{cite journal |vauthors= McDonough MA, Li V, Flashman E, Chowdhury R, Mohr C, Liénard BM, Zondlo J, Oldham NJ, Clifton IJ, Lewis J, McNeill LA, Kurzeja RJ, Hewitson KS, Yang E, Jordan S, Syed RS, Schofield CJ | title = Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2) | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 103 | issue = 26 | pages = 9814–9819 |date=June 2006 | pmid = 16782814 | doi = 10.1073/pnas.0601283103 | pmc=1502536| bibcode = 2006PNAS..103.9814M }}</ref><ref name="pmid19604478">{{cite journal |vauthors= Chowdhury R, McDonough MA, Mecinović J, Loenarz C, Flashman E, Hewitson KS, Domene C, Schofield CJ | title = Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases | journal = Structure | volume = 17 | issue = 7 | pages = 981–989 |date=July 2009 | pmid = 19604478 | doi = 10.1016/j.str.2009.06.002 }}</ref><ref name="pmid27561929">{{cite journal |vauthors= Chowdhury R, Leung IK, Tian YM, Abboud MI, Ge W, Domene C, Cantrelle FX, Landrieu I, Hardy AP, Pugh CW, Ratcliffe PJ, Claridge TD, Schofield CJ | title = Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases | journal = Nat. Commun. | volume = 7 | issue = | pages = 12673 |date=August 2016 | pmid = 27561929 | doi = 10.1038/ncomms12673 | pmc=5007464| bibcode = 2016NatCo...712673C }}</ref> a αKG-dependent dioxygenase that is involved in oxygen sensing,<ref name="pmid15581484">{{cite journal |vauthors=William C, Nicholls L, Ratcliffe P, Pugh C, Maxwell P | title = The prolyl hydroxylase enzymes that act as oxygen sensors regulating destruction of hypoxia-inducible factor α | journal = Advan. Enzyme Regul. | volume = 44 | issue = | pages = 75–92 | year = 2004 | pmid = 15581484 | pmc = | doi = 10.1016/j.advenzreg.2003.11.017 }}</ref> and [[isopenicillin N synthase]] (IPNS), a microbial αKG-dependent dioxygenase.<ref name="pmid9194566">{{cite journal |vauthors= Roach PL, Clifton IJ, Hensgens CM, Shibata N, Schofield CJ, Hajdu J, Baldwin JE | title = Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation | journal = Nature | volume = 387 | issue = 6635 | pages = 827–830 |date=June 1997 | pmid = 9194566 | doi = 10.1038/42990 | s2cid = 205032251 }}</ref>
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 [[EGLN1|prolyl hydroxylase isoform 2]] (PHD2),<ref name="pmid16782814">{{cite journal |vauthors= McDonough MA, Li V, Flashman E, Chowdhury R, Mohr C, Liénard BM, Zondlo J, Oldham NJ, Clifton IJ, Lewis J, McNeill LA, Kurzeja RJ, Hewitson KS, Yang E, Jordan S, Syed RS, [[Christopher J. Schofield|Schofield CJ]] | title = Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2) | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 103 | issue = 26 | pages = 9814–9819 |date=June 2006 | pmid = 16782814 | doi = 10.1073/pnas.0601283103 | pmc=1502536| bibcode = 2006PNAS..103.9814M | doi-access = free }}</ref><ref name="pmid19604478">{{cite journal |vauthors= Chowdhury R, McDonough MA, Mecinović J, Loenarz C, Flashman E, Hewitson KS, [[Carmen Domene|Domene C]], [[Christopher J. Schofield|Schofield CJ]] | title = Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases | journal = Structure | volume = 17 | issue = 7 | pages = 981–989 |date=July 2009 | pmid = 19604478 | doi = 10.1016/j.str.2009.06.002 | doi-access = free }}</ref><ref name="pmid27561929">{{cite journal |vauthors= Chowdhury R, Leung IK, Tian YM, Abboud MI, Ge W, [[Carmen Domene|Domene C]], Cantrelle FX, Landrieu I, Hardy AP, Pugh CW, Ratcliffe PJ, Claridge TD, [[Christopher J. Schofield|Schofield CJ]] | title = Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases | journal = Nat. Commun. | volume = 7 | pages = 12673 |date=August 2016 | pmid = 27561929 | doi = 10.1038/ncomms12673 | pmc=5007464| bibcode = 2016NatCo...712673C }}</ref> a αKG-dependent dioxygenase that is involved in oxygen sensing,<ref name="pmid15581484">{{cite journal |vauthors=William C, Nicholls L, Ratcliffe P, Pugh C, Maxwell P | title = The prolyl hydroxylase enzymes that act as oxygen sensors regulating destruction of hypoxia-inducible factor α | journal = Advan. Enzyme Regul. | volume = 44 | pages = 75–92 | year = 2004 | pmid = 15581484 | doi = 10.1016/j.advenzreg.2003.11.017 }}</ref> and [[isopenicillin N synthase]] (IPNS), a microbial αKG-dependent dioxygenase.<ref name="pmid9194566">{{cite journal |vauthors= Roach PL, Clifton IJ, Hensgens CM, Shibata N, [[Christopher J. Schofield|Schofield CJ]], Hajdu J, Baldwin JE | title = Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation | journal = Nature | volume = 387 | issue = 6635 | pages = 827–830 |date=June 1997 | pmid = 9194566 | doi = 10.1038/42990 | s2cid = 205032251 | doi-access = free }}</ref>


[[File:PHD2 iron binding site.png|thumb|center|upright=2|Simplified view of the active site of [[EGLN1|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 O<sub>2</sub>, are omitted for clarity.]]
[[File:PHD2 iron binding site.png|thumb|center|upright=2|Simplified view of the active site of [[EGLN1|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 O<sub>2</sub>, are omitted for clarity.]]


==Inhibitors==
==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|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).<ref name="pmid21390379 ">{{cite journal |vauthors= Rose NR, McDonough MA, King ON, Kawamura A, Schofield CJ | title = Inhibition of 2-oxoglutarate dependent oxygenases | journal = Chem. Soc. Rev. | volume = 40 | issue = 8 | pages = 4364–4397 |date=August 2011 | pmid = 21390379 | doi = 10.1039/c0cs00203h }}</ref><ref name="pmidChemSciNoPMID">{{cite journal |vauthors= Yeh TL, Leissing TM, Abboud MI, Thinnes CC, Atasoylu O, Holt-Martyn JP, Zhang D, Tumber A, Lippl K, Lohans CT, Leung IK, Morcrette H, Clifton IJ, Claridge TD, Kawamura A, Flashman E, Lu X, Ratcliffe PJ, Chowdhury R, Pugh CW, Schofield CJ | title = Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials | journal = Chem. Sci. | volume = 8 | issue = 11 | pages = 7651–7668 |date=September 2017 | pmid = 29435217| pmc = 5802278 | doi = 10.1039/C7SC02103H }}</ref> Although they are potent inhibitors of αKG-dependent dioxygenase, they lack selectivity and hence sometimes being referred to as so-called 'broad spectrum' inhibitors.<ref name="pmid26682036">{{cite journal |vauthors= Hopkinson RJ, Tumber A, Yapp C, Chowdhury R, Aik W, Che KH, Li XS, Kristensen JB, King ON, Chan MC, Yeoh KK, Choi H, Walport LJ, Thinnes CC, Bush JT, Lejeune C, Rydzik AM, Rose NR, Bagg EA, McDonough MA, Krojer T, Yue WW, Ng SS, Olsen L, Brennan PE, Oppermann U, Muller-Knapp S, Klose RJ, Ratcliffe PJ, Schofield CJ, Kawamura A | title = 5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation | journal = Chem. Sci. | volume = 4 | issue = 8 | pages = 3110–3117 |date=August 2013 | pmid = 26682036 | doi = 10.1039/C3SC51122G | pmc=4678600}}</ref> Inhibitors that compete against the substrate were also developed, such as peptidyl-based inhibitors that target human [[EGLN1|prolyl hydroxylase domain 2]] (PHD2)<ref name="pmid21665470 ">{{cite journal |vauthors= Kwon HS, Choi YK, Kim JW, Park YK, Yang EG, Ahn DR | title = Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides | journal = Bioorg. Med. Chem. Lett. | volume = 21 | issue = 14 | pages = 4325–4328 |date=July 2011 | pmid = 21665470 | doi = 10.1016/j.bmcl.2011.05.050 }}</ref> and [[Mildronate]], a drug molecule that is commonly used in Russia and Eastern Europe that target [[gamma-butyrobetaine dioxygenase]].<ref name="pmid16633095">{{cite journal | vauthors = Sesti C, Simkhovich BZ, Kalvinsh I, Kloner RA | title = Mildronate, a novel fatty acid oxidation inhibitor and antianginal agent, reduces myocardial infarct size without affecting hemodynamics | journal = Journal of Cardiovascular Pharmacology | volume = 47 | issue = 3 | pages = 493–9 | date = Mar 2006 | pmid = 16633095 | doi = 10.1097/01.fjc.0000211732.76668.d2 | doi-broken-date = 2020-09-10 }}</ref><ref name="pmid17204911">{{cite journal | vauthors = Liepinsh E, Vilskersts R, Loca D, Kirjanova O, Pugovichs O, Kalvinsh I, Dambrova M | title = Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction | journal = Journal of Cardiovascular Pharmacology | volume = 48 | issue = 6 | pages = 314–9 | date = Dec 2006 | pmid = 17204911 | doi = 10.1097/01.fjc.0000250077.07702.23 | s2cid = 1812127 }}</ref><ref name="pmid10812052">{{cite journal | vauthors = Hayashi Y, Kirimoto T, Asaka N, Nakano M, Tajima K, Miyake H, Matsuura N | title = Beneficial effects of MET-88, a gamma-butyrobetaine hydroxylase inhibitor in rats with heart failure following myocardial infarction | journal = European Journal of Pharmacology | volume = 395 | issue = 3 | pages = 217–24 | date = May 2000 | pmid = 10812052 | doi = 10.1016/S0014-2999(00)00098-4 }}</ref>
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|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).<ref name="pmid21390379 ">{{cite journal |vauthors= Rose NR, McDonough MA, King ON, Kawamura A, [[Christopher J. Schofield|Schofield CJ]] | title = Inhibition of 2-oxoglutarate dependent oxygenases | journal = Chem. Soc. Rev. | volume = 40 | issue = 8 | pages = 4364–4397 |date=August 2011 | pmid = 21390379 | doi = 10.1039/c0cs00203h }}</ref><ref name="pmidChemSciNoPMID">{{cite journal |vauthors= Yeh TL, Leissing TM, Abboud MI, Thinnes CC, Atasoylu O, Holt-Martyn JP, Zhang D, Tumber A, Lippl K, Lohans CT, Leung IK, Morcrette H, Clifton IJ, Claridge TD, Kawamura A, Flashman E, Lu X, Ratcliffe PJ, Chowdhury R, Pugh CW, [[Christopher J. Schofield|Schofield CJ]] | title = Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials | journal = Chem. Sci. | volume = 8 | issue = 11 | pages = 7651–7668 |date=September 2017 | pmid = 29435217| pmc = 5802278 | doi = 10.1039/C7SC02103H }}</ref> Although they are potent inhibitors of αKG-dependent dioxygenase, they lack selectivity and hence sometimes being referred to as so-called 'broad spectrum' inhibitors.<ref name="pmid26682036">{{cite journal |vauthors= Hopkinson RJ, Tumber A, Yapp C, Chowdhury R, Aik W, Che KH, Li XS, Kristensen JB, King ON, Chan MC, Yeoh KK, Choi H, Walport LJ, Thinnes CC, Bush JT, Lejeune C, Rydzik AM, Rose NR, Bagg EA, McDonough MA, Krojer T, Yue WW, Ng SS, Olsen L, Brennan PE, Oppermann U, Muller-Knapp S, Klose RJ, Ratcliffe PJ, [[Christopher J. Schofield|Schofield CJ]], Kawamura A | title = 5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation | journal = Chem. Sci. | volume = 4 | issue = 8 | pages = 3110–3117 |date=August 2013 | pmid = 26682036 | doi = 10.1039/C3SC51122G | pmc=4678600}}</ref> Inhibitors that compete against the substrate were also developed, such as peptidyl-based inhibitors that target human [[EGLN1|prolyl hydroxylase domain 2]] (PHD2)<ref name="pmid21665470 ">{{cite journal |vauthors= Kwon HS, Choi YK, Kim JW, Park YK, Yang EG, Ahn DR | title = Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides | journal = Bioorg. Med. Chem. Lett. | volume = 21 | issue = 14 | pages = 4325–4328 |date=July 2011 | pmid = 21665470 | doi = 10.1016/j.bmcl.2011.05.050 }}</ref> and [[Mildronate]], a drug molecule that is commonly used in Russia and Eastern Europe that target [[gamma-butyrobetaine dioxygenase]].<ref name="pmid16633095">{{cite journal | vauthors = Sesti C, Simkhovich BZ, Kalvinsh I, Kloner RA | title = Mildronate, a novel fatty acid oxidation inhibitor and antianginal agent, reduces myocardial infarct size without affecting hemodynamics | journal = Journal of Cardiovascular Pharmacology | volume = 47 | issue = 3 | pages = 493–9 | date = Mar 2006 | pmid = 16633095 | doi = 10.1097/01.fjc.0000211732.76668.d2 | s2cid = 45844835 | doi-access = free }}</ref><ref name="pmid17204911">{{cite journal | vauthors = Liepinsh E, Vilskersts R, Loca D, Kirjanova O, Pugovichs O, Kalvinsh I, Dambrova M | title = Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction | journal = Journal of Cardiovascular Pharmacology | volume = 48 | issue = 6 | pages = 314–9 | date = Dec 2006 | pmid = 17204911 | doi = 10.1097/01.fjc.0000250077.07702.23 | s2cid = 1812127 | doi-access = free }}</ref><ref name="pmid10812052">{{cite journal | vauthors = Hayashi Y, Kirimoto T, Asaka N, Nakano M, Tajima K, Miyake H, Matsuura N | title = Beneficial effects of MET-88, a gamma-butyrobetaine hydroxylase inhibitor in rats with heart failure following myocardial infarction | journal = European Journal of Pharmacology | volume = 395 | issue = 3 | pages = 217–24 | date = May 2000 | pmid = 10812052 | doi = 10.1016/S0014-2999(00)00098-4 }}</ref> Finally, as αKG-dependent dioxygenases require molecular oxygen as a co-substrate, it has also been shown that gaseous molecules such as carbon monoxide<ref name="doi101002cbic202100181">{{cite journal | vauthors = Mbenza NM, Nasarudin N, Vadakkedath PG, Patel K, Ismail AZ, Hanif M, Wright LJ, Sarojini V, Hartinger CG, Leung IK | title = Carbon monoxide is an inhibitor of HIF prolyl hydroxylase domain 2 | journal = ChemBioChem | volume = 22| issue = 15| pages = 2521–2525| date = June 2021 | pmid = 34137488| doi = 10.1002/cbic.202100181 | hdl = 11343/298654 | s2cid = 235460239 | hdl-access = free }}</ref> and nitric oxide<ref name="pmid12925778">{{cite journal | vauthors = Metzen E, Zhou J, Jelkmann W, Fandrey J, Brüne B | title = Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases | journal = Molecular Biology of the Cell | volume = 14 | issue = 8 | pages = 3470–3481 | date = August 2003 | pmid = 12925778 | doi = 10.1091/mbc.E02-12-0791 | pmc = 181582 }}</ref><ref name="pmid17060326">{{cite journal | vauthors = Berchner-Pfannschmidt U, Yamac H, Trinidad B, Fandrey J | title = Nitric oxide modulates oxygen sensing by hypoxia-inducible factor 1-dependent induction of prolyl hydroxylase 2 | journal = Journal of Biological Chemistry | volume = 282 | issue = 3 | pages = 1788–1796 | date = January 2007 | pmid = 17060326 | doi = 10.1074/jbc.M607065200 | pmc = | s2cid = 26185260 | doi-access = free }}</ref> are inhibitors of αKG-dependent dioxygenases, presumably by competing with molecular oxygen for the binding at the active site Fe(II) ion.


[[File:2OG Oxygenase Inhibitors.png|thumb|center|upright=4|Common inhibitors of αKG-dependent dioxygenases. They compete against the cosubstrate αKG for binding to the active site Fe(II).]]
[[File:2OG Oxygenase Inhibitors.png|thumb|center|upright=4|Common inhibitors of αKG-dependent dioxygenases. They compete against the cosubstrate αKG for binding to the active site Fe(II).]]


==Assays==
==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.<ref name="pmid28893416">{{cite journal |vauthors= Mbenza NM, Vadakkedath PG, McGillivray DJ, Leung IK | title = NMR studies of the non-haem Fe(II) and 2-oxoglutarate-dependent oxygenases | journal = J. Inorg. Biochem. | volume = 177 | issue = | pages =384–394 |date=December 2017 | pmid = 28893416 | doi = 10.1016/j.jinorgbio.2017.08.032 }}</ref> For example, assays were developed to study ligand binding,<ref name="pmid23234607">{{cite journal |vauthors= Leung IK, Demetriades M, Hardy AP, Lejeune C, Smart TJ, Szöllössi A, Kawamura A, Schofield CJ, Claridge TD | title = Reporter ligand NMR screening method for 2-oxoglutarate oxygenase inhibitors | journal = J. Med. Chem. | volume = 56 | issue = 2 | pages = 547–555 |date=January 2013 | pmid = 23234607 | doi = 10.1021/jm301583m | pmc=4673903}}</ref><ref name="pmid20025281">{{cite journal |vauthors= Leung IK, Flashman E, Yeoh KK, Schofield CJ, Claridge TD | title = Using NMR solvent water relaxation to investigate metalloenzyme-ligand binding interactions | journal = J. Med. Chem. | volume = 53 | issue = 2 | pages = 867–875 |date=January 2010 | pmid = 20025281 | doi = 10.1021/jm901537q }}</ref><ref name="pmidMedChemCommNoID">{{cite journal |vauthors= Khan A, Leśniak RK, Brem J, Rydzik AM, Choi H, Leung IK, McDonough MA, Schofield CJ, Claridge TD | title = Development and application of ligand-based NMR screening assays for γ-butyrobetaine hydroxylase | journal = Med. Chem. Commun. | volume = 7 | issue = 5 | pages = 873–880 |date=February 2017 | pmid = | doi = 10.1039/C6MD00004E }}</ref> enzyme kinetics,<ref name="pmid20095001">{{cite journal |vauthors= Hopkinson RJ, Hamed RB, Rose NR, Claridge TD, Schofield CJ | title = Monitoring the activity of 2-oxoglutarate dependent histone demethylases by NMR spectroscopy: direct observation of formaldehyde | journal = ChemBioChem | volume = 11 | issue = 4 | pages = 506–510 |date=March 2010 | pmid = 20095001 | doi = 10.1002/cbic.200900713 | s2cid = 42994868 }}</ref> modes of inhibition<ref name="pmid19886658">{{cite journal |vauthors= Poppe L, Tegley CM, Li V, Lewis J, Zondlo J, Yang E, Kurzeja RJ, Syed R | title = Different modes of inhibitor binding to prolyl hydroxylase by combined use of X-ray crystallography and NMR spectroscopy of paramagnetic complexes | journal = J. Am. Chem. Soc. | volume = 131 | issue = 46 | pages = 16654–16655 |date=November 2009 | pmid = 19886658 | doi = 10.1021/ja907933p }}</ref> as well as protein conformational change.<ref name="pmid18617893 ">{{cite journal |vauthors= Bleijlevens B, Shivarattan T, Flashman E, Yang Y, Simpson PJ, Koivisto P, Sedgwick B, Schofield CJ, Matthews SJ | title = Dynamic states of the DNA repair enzyme AlkB regulate product release | journal = EMBO Rep. | volume = 9 | issue = 9 | pages = 872–877 |date=September 2008 | pmid = 18617893 | doi = 10.1038/embor.2008.120 | pmc=2529343}}</ref> Mass spectrometry is also widely applied. It can be used to characterise enzyme kinetics,<ref name="pmid18063574 ">{{cite journal |vauthors= Flashman E, Bagg EA, Chowdhury R, Mecinović J, Loenarz C, McDonough MA, Hewitson KS, Schofield CJ | title = 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 | journal = J. Biol. Chem. | volume = 283 | issue = 7 | pages = 3808–3815 |date=February 2008 | pmid = 18063574 | doi = 10.1074/jbc.M707411200 | s2cid = 34893579 }}</ref> to guide enzyme inhibitor development,<ref name="pmid22639232 ">{{cite journal |vauthors= Demetriades M, Leung IK, Chowdhury R, Chan MC, McDonough MA, Yeoh KK, Tian YM, Claridge TD, Ratcliffe PJ, Woon EC, Schofield CJ| title = Dynamic combinatorial chemistry employing boronic acids/boronate esters leads to potent oxygenase inhibitors | journal = Angew. Chem. Int. Ed. | volume = 51 | issue = 27 | pages = 6672–6675 |date=July 2012 | pmid = 22639232 | doi = 10.1002/anie.201202000 }}</ref> study ligand and metal binding<ref name="pmid18058781">{{cite journal |vauthors= Mecinović J, Chowdhury R, Liénard BM, Flashman E, Buck MR, Oldham NJ, Schofield CJ | title = ESI-MS studies on prolyl hydroxylase domain 2 reveal a new metal binding site | journal = ChemMedChem | volume = 3 | issue = 4 | pages = 569–572 |date=April 2008 | pmid = 18058781 | doi = 10.1002/cmdc.200700233 }}</ref> as well as analyse protein conformational change.<ref name="pmid19364117">{{cite journal |vauthors= Stubbs CJ, Loenarz C, Mecinović J, Yeoh KK, Hindley N, Liénard BM, Sobott F, Schofield CJ, Flashman E | title = Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure | journal = J. Med. Chem. | volume = 52 | issue = 9 | pages = 2799–2805 |date=May 2009 | pmid = 19364117 | doi = 10.1021/jm900285r }}</ref> Assays using spectrophotometry were also used,<ref name="pmid 27812832 ">{{cite journal |vauthors= Proshlyakov DA, McCracken J, Hausinger RP | title = Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study | journal = J. Biol. Inorg. Chem. | volume = 22 | issue = 2–3 | pages = 367–379 |date=April 2016 | pmid = 27812832 | doi = 10.1007/s00775-016-1406-3 | pmc=5352539}}</ref> for example those that measure 2OG oxidation,<ref name="pmid15582567">{{cite journal |vauthors= McNeill LA, Bethge L, Hewitson KS, Schofield CJ| title = A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases | journal = Anal. Biochem. | volume = 336 | issue = 1 | pages = 125–131 |date=January 2005 | pmid = 15582567 | doi = 10.1016/j.ab.2004.09.019 }}</ref> co-product succinate formation<ref name="pmid16643838">{{cite journal |vauthors= Luo L, Pappalardi MB, Tummino PJ, Copeland RA, Fraser ME, Grzyska PK, Hausinger RP | title = An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation | journal = Anal. Biochem. | volume = 353 | issue = 1 | pages = 69–74 |date=June 2006 | pmid = 16643838 | doi = 10.1016/j.ab.2006.03.033 }}</ref> or product formation.<ref name="pmid22730246">{{cite journal |vauthors= Rydzik AM, Leung IK, Kochan GT, Thalhammer A, Oppermann U, Claridge TD, Schofield CJ | title = Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase | journal = ChemBioChem | volume = 13 | issue = 11 | pages = 1559–1563 |date=July 2012 | pmid = 22730246 | doi = 10.1002/cbic.201200256 | s2cid = 13956474 }}</ref> Other biophysical techniques including (but not limited to) isothermal titration calorimetry (ITC)<ref name="pmid26936969">{{cite journal |vauthors= Huang CW, Liu HC, Shen CP, Chen YT, Lee SJ, Lloyd MD, Lee HJ | title = The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase | journal = Biochem. J. | volume = 473 | issue = 9 | pages = 1179–1189 |date=May 2016 | pmid = 26936969 | doi = 10.1042/BCJ20160146 | url = http://opus.bath.ac.uk/49739/1/Supp_Fig_all.pdf }}</ref> and electron paramagnetic resonance (EPR) were also applied.<ref name="pmid 22687491 ">{{cite journal |vauthors= Flagg SC, Martin CB, Taabazuing CY, Holmes BE, Knapp MJ | title = Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH) | journal = J. Inorg. Biochem. | volume = 113 | issue = | pages = 25–30 |date=August 2012 | pmid = 22687491 | doi = 10.1016/j.jinorgbio.2012.03.002 | pmc=3525482}}</ref> Radioactive assays that uses <sup>14</sup>C labelled substrates were also developed and used.<ref name="pmid3028379">{{cite journal |vauthors= Cunliffe CJ, Franklin TJ, Gaskell RM | title = Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns | journal = Biochem. J. | volume = 240 | issue = 2 | pages = 617–619 |date=December 1986 | pmid = 3028379 | doi = 10.1042/bj2400617 | pmc = 1147460 }}</ref> Given αKG-dependent dioxygenases require oxygen for their catalytic activity, oxygen consumption assay was also applied.<ref name="pmid16952279">{{cite journal |vauthors= Ehrismann D, Flashman E, Genn DN, Mathioudakis N, Hewitson KS, Ratcliffe PJ, Schofield CJ| title = Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay | journal = Biochem. J. | volume = 401 | issue = 1 | pages = 227–234 |date=January 2007 | pmid = 16952279 | doi = 10.1042/BJ20061151 | pmc=1698668}}</ref>
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.<ref name="pmid28893416">{{cite journal |vauthors= Mbenza NM, Vadakkedath PG, McGillivray DJ, Leung IK | title = NMR studies of the non-haem Fe(II) and 2-oxoglutarate-dependent oxygenases | journal = J. Inorg. Biochem. | volume = 177 | pages =384–394 |date=December 2017 | pmid = 28893416 | doi = 10.1016/j.jinorgbio.2017.08.032 }}</ref> For example, assays were developed to study ligand binding,<ref name="pmid23234607">{{cite journal |vauthors= Leung IK, Demetriades M, Hardy AP, Lejeune C, Smart TJ, Szöllössi A, Kawamura A, [[Christopher J. Schofield|Schofield CJ]], Claridge TD | title = Reporter ligand NMR screening method for 2-oxoglutarate oxygenase inhibitors | journal = J. Med. Chem. | volume = 56 | issue = 2 | pages = 547–555 |date=January 2013 | pmid = 23234607 | doi = 10.1021/jm301583m | pmc=4673903}}</ref><ref name="pmid20025281">{{cite journal |vauthors= Leung IK, Flashman E, Yeoh KK, [[Christopher J. Schofield|Schofield CJ]], Claridge TD | title = Using NMR solvent water relaxation to investigate metalloenzyme-ligand binding interactions | journal = J. Med. Chem. | volume = 53 | issue = 2 | pages = 867–875 |date=January 2010 | pmid = 20025281 | doi = 10.1021/jm901537q }}</ref><ref name="pmidMedChemCommNoID">{{cite journal |vauthors= Khan A, Leśniak RK, Brem J, Rydzik AM, Choi H, Leung IK, McDonough MA, [[Christopher J. Schofield|Schofield CJ]], Claridge TD | title = Development and application of ligand-based NMR screening assays for γ-butyrobetaine hydroxylase | journal = Med. Chem. Commun. | volume = 7 | issue = 5 | pages = 873–880 |date=February 2017 | doi = 10.1039/C6MD00004E | doi-access = free | hdl = 2292/30083 | hdl-access = free }}</ref> enzyme kinetics,<ref name="pmid20095001">{{cite journal |vauthors= Hopkinson RJ, Hamed RB, Rose NR, Claridge TD, [[Christopher J. Schofield|Schofield CJ]] | title = Monitoring the activity of 2-oxoglutarate dependent histone demethylases by NMR spectroscopy: direct observation of formaldehyde | journal = ChemBioChem | volume = 11 | issue = 4 | pages = 506–510 |date=March 2010 | pmid = 20095001 | doi = 10.1002/cbic.200900713 | s2cid = 42994868 }}</ref> modes of inhibition<ref name="pmid19886658">{{cite journal |vauthors= Poppe L, Tegley CM, Li V, Lewis J, Zondlo J, Yang E, Kurzeja RJ, Syed R | title = Different modes of inhibitor binding to prolyl hydroxylase by combined use of X-ray crystallography and NMR spectroscopy of paramagnetic complexes | journal = J. Am. Chem. Soc. | volume = 131 | issue = 46 | pages = 16654–16655 |date=November 2009 | pmid = 19886658 | doi = 10.1021/ja907933p | url = https://figshare.com/articles/journal_contribution/2810647 }}</ref> as well as protein conformational change.<ref name="pmid18617893 ">{{cite journal |vauthors= Bleijlevens B, Shivarattan T, Flashman E, Yang Y, Simpson PJ, Koivisto P, Sedgwick B, [[Christopher J. Schofield|Schofield CJ]], Matthews SJ | title = Dynamic states of the DNA repair enzyme AlkB regulate product release | journal = EMBO Rep. | volume = 9 | issue = 9 | pages = 872–877 |date=September 2008 | pmid = 18617893 | doi = 10.1038/embor.2008.120 | pmc=2529343}}</ref> Mass spectrometry is also widely applied. It can be used to characterise enzyme kinetics,<ref name="pmid18063574 ">{{cite journal |vauthors= Flashman E, Bagg EA, Chowdhury R, Mecinović J, Loenarz C, McDonough MA, Hewitson KS, Schofield CJ | title = 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 | journal = J. Biol. Chem. | volume = 283 | issue = 7 | pages = 3808–3815 |date=February 2008 | pmid = 18063574 | doi = 10.1074/jbc.M707411200 | s2cid = 34893579 | doi-access = free }}</ref> to guide enzyme inhibitor development,<ref name="pmid22639232 ">{{cite journal |vauthors= Demetriades M, Leung IK, Chowdhury R, Chan MC, McDonough MA, Yeoh KK, Tian YM, Claridge TD, Ratcliffe PJ, Woon EC, Schofield CJ| title = Dynamic combinatorial chemistry employing boronic acids/boronate esters leads to potent oxygenase inhibitors | journal = Angew. Chem. Int. Ed. | volume = 51 | issue = 27 | pages = 6672–6675 |date=July 2012 | pmid = 22639232 | doi = 10.1002/anie.201202000 }}</ref> study ligand and metal binding<ref name="pmid18058781">{{cite journal |vauthors= Mecinović J, Chowdhury R, Liénard BM, Flashman E, Buck MR, Oldham NJ, Schofield CJ | title = ESI-MS studies on prolyl hydroxylase domain 2 reveal a new metal binding site | journal = ChemMedChem | volume = 3 | issue = 4 | pages = 569–572 |date=April 2008 | pmid = 18058781 | doi = 10.1002/cmdc.200700233 | s2cid = 37628097 }}</ref> as well as analyse protein conformational change.<ref name="pmid19364117">{{cite journal |vauthors= Stubbs CJ, Loenarz C, Mecinović J, Yeoh KK, Hindley N, Liénard BM, Sobott F, Schofield CJ, Flashman E | title = Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure | journal = J. Med. Chem. | volume = 52 | issue = 9 | pages = 2799–2805 |date=May 2009 | pmid = 19364117 | doi = 10.1021/jm900285r }}</ref> Assays using spectrophotometry were also used,<ref name="pmid 27812832 ">{{cite journal |vauthors= Proshlyakov DA, McCracken J, Hausinger RP | title = Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study | journal = J. Biol. Inorg. Chem. | volume = 22 | issue = 2–3 | pages = 367–379 |date=April 2016 | pmid = 27812832 | doi = 10.1007/s00775-016-1406-3 | pmc=5352539}}</ref> for example those that measure 2OG oxidation,<ref name="pmid15582567">{{cite journal |vauthors= McNeill LA, Bethge L, Hewitson KS, Schofield CJ| title = A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases | journal = Anal. Biochem. | volume = 336 | issue = 1 | pages = 125–131 |date=January 2005 | pmid = 15582567 | doi = 10.1016/j.ab.2004.09.019 }}</ref> co-product succinate formation<ref name="pmid16643838">{{cite journal |vauthors= Luo L, Pappalardi MB, Tummino PJ, Copeland RA, Fraser ME, Grzyska PK, Hausinger RP | title = An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation | journal = Anal. Biochem. | volume = 353 | issue = 1 | pages = 69–74 |date=June 2006 | pmid = 16643838 | doi = 10.1016/j.ab.2006.03.033 }}</ref> or product formation.<ref name="pmid22730246">{{cite journal |vauthors= Rydzik AM, Leung IK, Kochan GT, Thalhammer A, Oppermann U, Claridge TD, Schofield CJ | title = Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase | journal = ChemBioChem | volume = 13 | issue = 11 | pages = 1559–1563 |date=July 2012 | pmid = 22730246 | doi = 10.1002/cbic.201200256 | s2cid = 13956474 }}</ref> Other biophysical techniques including (but not limited to) isothermal titration calorimetry (ITC)<ref name="pmid26936969">{{cite journal |vauthors= Huang CW, Liu HC, Shen CP, Chen YT, Lee SJ, Lloyd MD, Lee HJ | title = The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase | journal = Biochem. J. | volume = 473 | issue = 9 | pages = 1179–1189 |date=May 2016 | pmid = 26936969 | doi = 10.1042/BCJ20160146 | url = http://opus.bath.ac.uk/49739/1/Supp_Fig_all.pdf }}</ref> and electron paramagnetic resonance (EPR) were also applied.<ref name="pmid 22687491 ">{{cite journal |vauthors= Flagg SC, Martin CB, Taabazuing CY, Holmes BE, Knapp MJ | title = Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH) | journal = J. Inorg. Biochem. | volume = 113 | pages = 25–30 |date=August 2012 | pmid = 22687491 | doi = 10.1016/j.jinorgbio.2012.03.002 | pmc=3525482}}</ref> Radioactive assays that uses <sup>14</sup>C labelled substrates were also developed and used.<ref name="pmid3028379">{{cite journal |vauthors= Cunliffe CJ, Franklin TJ, Gaskell RM | title = Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns | journal = Biochem. J. | volume = 240 | issue = 2 | pages = 617–619 |date=December 1986 | pmid = 3028379 | doi = 10.1042/bj2400617 | pmc = 1147460 }}</ref> Given αKG-dependent dioxygenases require oxygen for their catalytic activity, oxygen consumption assay was also applied.<ref name="pmid16952279">{{cite journal |vauthors= Ehrismann D, Flashman E, Genn DN, Mathioudakis N, Hewitson KS, Ratcliffe PJ, Schofield CJ| title = Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay | journal = Biochem. J. | volume = 401 | issue = 1 | pages = 227–234 |date=January 2007 | pmid = 16952279 | doi = 10.1042/BJ20061151 | pmc=1698668}}</ref>


==Further reading==<!--dated primary literature-->
==Further reading==<!--dated primary literature-->
*{{Cite journal|last1=Martinez|first1=Salette|last2=Hausinger|first2=Robert P.|date=2015-08-21|title=Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases|journal=The Journal of Biological Chemistry |volume=290|issue=34|pages=20702–20711|doi=10.1074/jbc.R115.648691|issn=0021-9258|pmc=4543632|pmid=26152721}}
*{{Cite journal|last1=Martinez|first1=Salette|last2=Hausinger|first2=Robert P.|date=2015-08-21|title=Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases|journal=The Journal of Biological Chemistry |volume=290|issue=34|pages=20702–20711|doi=10.1074/jbc.R115.648691|issn=0021-9258|pmc=4543632|pmid=26152721|doi-access=free}}
*{{cite journal |vauthors= Hegg EL, Que L Jr | title = The 2-His-1-carboxylate facial triad--an emerging structural motif in mononuclear non-heme iron(II) enzymes | journal = Eur. J. Biochem. | volume = 250 | issue = 3 | pages = 625–629 |date=December 1997 | pmid = 9461283 | doi = 10.1111/j.1432-1033.1997.t01-1-00625.x }}.
*{{cite journal |vauthors= Hegg EL, Que L Jr | title = The 2-His-1-carboxylate facial triad--an emerging structural motif in mononuclear non-heme iron(II) enzymes | journal = Eur. J. Biochem. | volume = 250 | issue = 3 | pages = 625–629 |date=December 1997 | pmid = 9461283 | doi = 10.1111/j.1432-1033.1997.t01-1-00625.x | doi-access = free }}.
*{{cite journal |vauthors= Myllylä R, Tuderman L, Kivirikko KI | title = Mechanism of the prolyl hydroxylase reaction. 2. Kinetic analysis of the reaction sequence | journal = Eur. J. Biochem. | volume = 80 | issue = 2 | pages = 349–357 |date=November 1977 | pmid = 200425 | doi = 10.1111/j.1432-1033.1977.tb11889.x }}
*{{cite journal |vauthors= Myllylä R, Tuderman L, Kivirikko KI | title = Mechanism of the prolyl hydroxylase reaction. 2. Kinetic analysis of the reaction sequence | journal = Eur. J. Biochem. | volume = 80 | issue = 2 | pages = 349–357 |date=November 1977 | pmid = 200425 | doi = 10.1111/j.1432-1033.1977.tb11889.x | doi-access = free }}
*{{cite journal |vauthors= Valegård K, Terwisscha van Scheltinga AC, Dubus A, Ranghino G, Oster LM, Hajdu J, Andersson I | title = The structural basis of cephalosporin formation in a mononuclear ferrous enzyme | journal = Nat. Struct. Mol. Biol. | volume = 11 | issue = 1 | pages = 95–101 |date=January 2004 | pmid = 14718929 | doi = 10.1038/nsmb712 | s2cid = 1205987 | url = https://pure.rug.nl/ws/files/14617546/2004NatureStructMolBiolValegardSupp3.pdf }}
*{{cite journal |vauthors= Valegård K, Terwisscha van Scheltinga AC, Dubus A, Ranghino G, Oster LM, Hajdu J, Andersson I | title = The structural basis of cephalosporin formation in a mononuclear ferrous enzyme | journal = Nat. Struct. Mol. Biol. | volume = 11 | issue = 1 | pages = 95–101 |date=January 2004 | pmid = 14718929 | doi = 10.1038/nsmb712 | s2cid = 1205987 | url = https://pure.rug.nl/ws/files/14617546/2004NatureStructMolBiolValegardSupp3.pdf }}
*{{cite journal |vauthors= Price JC, Barr EW, Tirupati B, Bollinger JM Jr, Krebs C | title = The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli | journal = Biochemistry | volume = 42 | issue = 24 | pages = 7497–7508 |date=June 2003 | pmid = 12809506 | doi = 10.1021/bi030011f }}
*{{cite journal |vauthors= Price JC, Barr EW, Tirupati B, Bollinger JM Jr, Krebs C | title = The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli | journal = Biochemistry | volume = 42 | issue = 24 | pages = 7497–7508 |date=June 2003 | pmid = 12809506 | doi = 10.1021/bi030011f }}
*{{cite journal |vauthors= Proshlyakov DA, Henshaw TF, Monterosso GR, Ryle MJ, Hausinger RP | title = Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase | journal = J. Am. Chem. Soc. | volume = 126 | issue = 4 | pages = 1022–1023 |date=February 2004 | pmid = 14746461 | doi = 10.1021/ja039113j }}
*{{cite journal |vauthors= Proshlyakov DA, Henshaw TF, Monterosso GR, Ryle MJ, Hausinger RP | title = Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase | journal = J. Am. Chem. Soc. | volume = 126 | issue = 4 | pages = 1022–1023 |date=February 2004 | pmid = 14746461 | doi = 10.1021/ja039113j }}
*{{cite journal |vauthors= Hewitson KS, Granatino N, Welford RW, McDonough MA, Schofield CJ | title = Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling | journal = Phil. Trans. R. Soc. A | volume = 363 | issue = 1829 | pages = 807–828 |date=April 2005 | pmid = 15901537 | doi = 10.1098/rsta.2004.1540 | bibcode = 2005RSPTA.363..807H | s2cid = 8568103 }}
*{{cite journal |vauthors= Hewitson KS, Granatino N, Welford RW, McDonough MA, Schofield CJ | title = Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling | journal = Phil. Trans. R. Soc. A | volume = 363 | issue = 1829 | pages = 807–828 |date=April 2005 | pmid = 15901537 | doi = 10.1098/rsta.2004.1540 | bibcode = 2005RSPTA.363..807H | s2cid = 8568103 }}
*{{cite journal |vauthors= Wick CR, Lanig H, Jäger CM, Burzlaff N, Clark T | title = Structural Insight into the Prolyl Hydroxylase PHD2: A Molecular Dynamics and DFT Study | journal = Eur. J. Inorg. Chem. | volume = 2012 | issue = 31| pages = 4973–4985 |date=November 2012 | pmid = | doi = 10.1002/ejic.201200391 }}
*{{cite journal |vauthors= Wick CR, Lanig H, Jäger CM, Burzlaff N, Clark T | title = Structural Insight into the Prolyl Hydroxylase PHD2: A Molecular Dynamics and DFT Study | journal = Eur. J. Inorg. Chem. | volume = 2012 | issue = 31| pages = 4973–4985 |date=November 2012 | doi = 10.1002/ejic.201200391 | doi-access = free }}


==References==
==References==

Latest revision as of 06:03, 27 May 2024

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

[edit]

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

[edit]

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

[edit]

Protein

[edit]

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

[edit]

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

[edit]

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

[edit]

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

[edit]

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

[edit]
  • Martinez, Salette; Hausinger, Robert P. (2015-08-21). "Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases". The Journal of Biological Chemistry. 290 (34): 20702–20711. doi:10.1074/jbc.R115.648691. ISSN 0021-9258. PMC 4543632. PMID 26152721.
  • Hegg EL, Que L Jr (December 1997). "The 2-His-1-carboxylate facial triad--an emerging structural motif in mononuclear non-heme iron(II) enzymes". Eur. J. Biochem. 250 (3): 625–629. doi:10.1111/j.1432-1033.1997.t01-1-00625.x. PMID 9461283..
  • Myllylä R, Tuderman L, Kivirikko KI (November 1977). "Mechanism of the prolyl hydroxylase reaction. 2. Kinetic analysis of the reaction sequence". Eur. J. Biochem. 80 (2): 349–357. doi:10.1111/j.1432-1033.1977.tb11889.x. PMID 200425.
  • Valegård K, Terwisscha van Scheltinga AC, Dubus A, Ranghino G, Oster LM, Hajdu J, Andersson I (January 2004). "The structural basis of cephalosporin formation in a mononuclear ferrous enzyme" (PDF). Nat. Struct. Mol. Biol. 11 (1): 95–101. doi:10.1038/nsmb712. PMID 14718929. S2CID 1205987.
  • Price JC, Barr EW, Tirupati B, Bollinger JM Jr, Krebs C (June 2003). "The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli". Biochemistry. 42 (24): 7497–7508. doi:10.1021/bi030011f. PMID 12809506.
  • Proshlyakov DA, Henshaw TF, Monterosso GR, Ryle MJ, Hausinger RP (February 2004). "Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase". J. Am. Chem. Soc. 126 (4): 1022–1023. doi:10.1021/ja039113j. PMID 14746461.
  • Hewitson KS, Granatino N, Welford RW, McDonough MA, Schofield CJ (April 2005). "Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling". Phil. Trans. R. Soc. A. 363 (1829): 807–828. Bibcode:2005RSPTA.363..807H. doi:10.1098/rsta.2004.1540. PMID 15901537. S2CID 8568103.
  • Wick CR, Lanig H, Jäger CM, Burzlaff N, Clark T (November 2012). "Structural Insight into the Prolyl Hydroxylase PHD2: A Molecular Dynamics and DFT Study". Eur. J. Inorg. Chem. 2012 (31): 4973–4985. doi:10.1002/ejic.201200391.

References

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