Alpha-ketoglutarate-dependent hydroxylases

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 pathways.[6][7][8] In plants, αKG-dependent dioxygenases are involved in diverse reactions in plant metabolism.[9] These include flavonoid biosynthesis,[10] and ethylene biosyntheses.[11] In mammals and humans, αKG-dependent dioxygenase have functional roles in biosyntheses (e.g. collagen biosynthesis[12] and L-carnitine biosynthesis[13]), post-translational modifications (e.g. protein hydroxylation[14]), epigenetic regulations (e.g. histone and DNA demethylation[15]), as well as sensors of energy metabolism.[16]

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.[17][18]

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:[19][20]

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.[21]

Consensus catalytic mechanism of the αKG-dependent dioxygenase superfamily.

Structure

Protein

All αKG-dependent dioxygenases contain a conserved double-stranded β-helix (DSBH, also known as cupin) fold, which is formed with two β-sheets.[22][23]

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.[24]

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),[25][26][27] a αKG-dependent dioxygenase that is involved in oxygen sensing,[28] and isopenicillin N synthase (IPNS), a microbial αKG-dependent dioxygenase.[29]

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

Inhibitors

Given the important biological roles that αKG-dependent dioxygenase play, many αKG-dependent dioxygenase inhibitors were developed. The inhibitors that were regularly used to target αKG-dependent dioxygenase include N-oxalylglycine (NOG), pyridine-2,4-dicarboxylic acid (2,4-PDCA), 5-carboxy-8-hydroxyquinoline, FG-2216 and FG-4592, which were all designed mimic the co-substrate αKG and compete against the binding of αKG at the enzyme active site Fe(II).[30][31] Although they are potent inhibitors of αKG-dependent dioxygenase, they lack selectivity and hence sometimes being referred to as so-called 'broad spectrum' inhibitors.[32] Inhibitors that compete against the substrate were also developed, such as peptidyl-based inhibitors that target human prolyl hydroxylase domain 2 (PHD2)[33] and Mildronate, a drug molecule that is commonly used in Russia and Eastern Europe that target gamma-butyrobetaine dioxygenase.[34][35][36]

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

Assays

Many assays were developed to study αKG-dependent dioxygenases so that information such as enzyme kinetics, enzyme inhibition and ligand binding can be obtained. Nuclear magnetic resonance (NMR) spectroscopy is widely applied to study αKG-dependent dioxygenases.[37] For example, assays were developed to study ligand binding,[38][39][40] enzyme kinetics,[41] modes of inhibition[42] as well as protein conformational change.[43] Mass spectrometry is also widely applied. It can be used to characterise enzyme kinetics,[44] to guide enzyme inhibitor development,[45] study ligand and metal binding[46] as well as analyse protein conformational change.[47] Assays using spectrophotometry were also used,[48] for example those that measure 2OG oxidation,[49] co-product succinate formation[50] or product formation.[51] Other biophysical techniques including (but not limited to) isothermal titration calorimetry (ITC)[52] and electron paramagnetic resonance (EPR) were also applied.[53] Radioactive assays that uses 14C labelled substrates were also developed and used.[54] Given αKG-dependent dioxygenases require oxygen for their catalytic activity, oxygen consumption assay was also applied.[55]

Further reading
  • 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.

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