Alpha-ketoglutarate-dependent hydroxylases explained

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

Biological function

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

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

Catalytic mechanism

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

R3CH +

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

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

Alternative mechanisms have failed to gain support.[23]

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]

Further reading

Notes and References

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