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]
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]
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]
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.
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]
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.
PMID12809506.
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.
PMID14746461.
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.
PMID15901537.
S2CID8568103.
^Prescott AG, Lloyd MD (August 2000). "The iron(II) and 2-oxoacid-dependent dioxygenases and their role in metabolism". Nat. Prod. Rep. 17 (4): 367–383.
doi:
10.1039/A902197C.
PMID11014338.
^Loenarz C,
Schofield CJ (January 2011). "Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases". Trends Biochem. Sci. 36 (1): 7–18.
doi:
10.1016/j.tibs.2010.07.002.
PMID20728359.
^Tarhonskaya H, Szöllössi A, Leung IK, Bush JT, Henry L, Chowdhury R, Iqbal A, Claridge TD,
Schofield CJ, Flashman E (April 2014). "Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases". Biochemistry. 53 (15): 2483–2493.
doi:
10.1021/bi500086p.
PMID24684493.
^McDonough MA, Loenarz C, Chowdhury R, Clifton IJ,
Schofield CJ (December 2010). "Structural studies on human 2-oxoglutarate dependent oxygenases". Curr. Opin. Struct. Biol. 20 (6): 659–672.
doi:
10.1016/j.sbi.2010.08.006.
PMID20888218.
^Clifton IJ, McDonough MA, Ehrismann D, Kershaw NJ, Granatino N,
Schofield CJ (April 2006). "Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins". J. Inorg. Biochem. 100 (4): 644–669.
doi:
10.1016/j.jinorgbio.2006.01.024.
PMID16513174.
^Kwon HS, Choi YK, Kim JW, Park YK, Yang EG, Ahn DR (July 2011). "Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides". Bioorg. Med. Chem. Lett. 21 (14): 4325–4328.
doi:
10.1016/j.bmcl.2011.05.050.
PMID21665470.
^Hayashi Y, Kirimoto T, Asaka N, Nakano M, Tajima K, Miyake H, Matsuura N (May 2000). "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.
doi:
10.1016/S0014-2999(00)00098-4.
PMID10812052.
^Mbenza NM, Nasarudin N, Vadakkedath PG, Patel K, Ismail AZ, Hanif M, Wright LJ, Sarojini V, Hartinger CG, Leung IK (June 2021). "Carbon monoxide is an inhibitor of HIF prolyl hydroxylase domain 2". ChemBioChem. 22 (15): 2521–2525.
doi:
10.1002/cbic.202100181.
hdl:11343/298654.
PMID34137488.
S2CID235460239.
^Hopkinson RJ, Hamed RB, Rose NR, Claridge TD,
Schofield CJ (March 2010). "Monitoring the activity of 2-oxoglutarate dependent histone demethylases by NMR spectroscopy: direct observation of formaldehyde". ChemBioChem. 11 (4): 506–510.
doi:
10.1002/cbic.200900713.
PMID20095001.
S2CID42994868.
^McNeill LA, Bethge L, Hewitson KS, Schofield CJ (January 2005). "A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases". Anal. Biochem. 336 (1): 125–131.
doi:
10.1016/j.ab.2004.09.019.
PMID15582567.
^Luo L, Pappalardi MB, Tummino PJ, Copeland RA, Fraser ME, Grzyska PK, Hausinger RP (June 2006). "An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation". Anal. Biochem. 353 (1): 69–74.
doi:
10.1016/j.ab.2006.03.033.
PMID16643838.
^Rydzik AM, Leung IK, Kochan GT, Thalhammer A, Oppermann U, Claridge TD, Schofield CJ (July 2012). "Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase". ChemBioChem. 13 (11): 1559–1563.
doi:
10.1002/cbic.201200256.
PMID22730246.
S2CID13956474.
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]
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]
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]
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.
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]
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.
PMID12809506.
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.
PMID14746461.
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.
PMID15901537.
S2CID8568103.
^Prescott AG, Lloyd MD (August 2000). "The iron(II) and 2-oxoacid-dependent dioxygenases and their role in metabolism". Nat. Prod. Rep. 17 (4): 367–383.
doi:
10.1039/A902197C.
PMID11014338.
^Loenarz C,
Schofield CJ (January 2011). "Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases". Trends Biochem. Sci. 36 (1): 7–18.
doi:
10.1016/j.tibs.2010.07.002.
PMID20728359.
^Tarhonskaya H, Szöllössi A, Leung IK, Bush JT, Henry L, Chowdhury R, Iqbal A, Claridge TD,
Schofield CJ, Flashman E (April 2014). "Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases". Biochemistry. 53 (15): 2483–2493.
doi:
10.1021/bi500086p.
PMID24684493.
^McDonough MA, Loenarz C, Chowdhury R, Clifton IJ,
Schofield CJ (December 2010). "Structural studies on human 2-oxoglutarate dependent oxygenases". Curr. Opin. Struct. Biol. 20 (6): 659–672.
doi:
10.1016/j.sbi.2010.08.006.
PMID20888218.
^Clifton IJ, McDonough MA, Ehrismann D, Kershaw NJ, Granatino N,
Schofield CJ (April 2006). "Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins". J. Inorg. Biochem. 100 (4): 644–669.
doi:
10.1016/j.jinorgbio.2006.01.024.
PMID16513174.
^Kwon HS, Choi YK, Kim JW, Park YK, Yang EG, Ahn DR (July 2011). "Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides". Bioorg. Med. Chem. Lett. 21 (14): 4325–4328.
doi:
10.1016/j.bmcl.2011.05.050.
PMID21665470.
^Hayashi Y, Kirimoto T, Asaka N, Nakano M, Tajima K, Miyake H, Matsuura N (May 2000). "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.
doi:
10.1016/S0014-2999(00)00098-4.
PMID10812052.
^Mbenza NM, Nasarudin N, Vadakkedath PG, Patel K, Ismail AZ, Hanif M, Wright LJ, Sarojini V, Hartinger CG, Leung IK (June 2021). "Carbon monoxide is an inhibitor of HIF prolyl hydroxylase domain 2". ChemBioChem. 22 (15): 2521–2525.
doi:
10.1002/cbic.202100181.
hdl:11343/298654.
PMID34137488.
S2CID235460239.
^Hopkinson RJ, Hamed RB, Rose NR, Claridge TD,
Schofield CJ (March 2010). "Monitoring the activity of 2-oxoglutarate dependent histone demethylases by NMR spectroscopy: direct observation of formaldehyde". ChemBioChem. 11 (4): 506–510.
doi:
10.1002/cbic.200900713.
PMID20095001.
S2CID42994868.
^McNeill LA, Bethge L, Hewitson KS, Schofield CJ (January 2005). "A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases". Anal. Biochem. 336 (1): 125–131.
doi:
10.1016/j.ab.2004.09.019.
PMID15582567.
^Luo L, Pappalardi MB, Tummino PJ, Copeland RA, Fraser ME, Grzyska PK, Hausinger RP (June 2006). "An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation". Anal. Biochem. 353 (1): 69–74.
doi:
10.1016/j.ab.2006.03.033.
PMID16643838.
^Rydzik AM, Leung IK, Kochan GT, Thalhammer A, Oppermann U, Claridge TD, Schofield CJ (July 2012). "Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase". ChemBioChem. 13 (11): 1559–1563.
doi:
10.1002/cbic.201200256.
PMID22730246.
S2CID13956474.