Alpha-ketoglutarate-dependent hydroxylases are a major class of
non-heme iron protein In biochemistry, non-heme iron proteins describe families of enzymes that utilize iron at the active site but lack heme cofactors. Iron-sulfur proteins, including those that are enzymes, are not included in this definition.
Some of non-heme iron p ...
s that catalyse a wide range of reactions. These reactions include hydroxylation reactions, demethylations, ring expansions, ring closures, and desaturations.
Functionally, the αKG-dependent hydroxylases are comparable to
cytochrome P450
Cytochromes P450 (CYPs) are a superfamily of enzymes containing heme as a cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various co ...
enzymes. Both use O
2 and reducing equivalents as cosubstrates and both generate water.
Biological function
αKG-dependent hydroxylases have diverse roles.
In microorganisms such as bacteria, αKG-dependent dioxygenases are involved in many biosynthetic and metabolic pathways;
for example, in
''E. coli'', the
AlkB
AlkB (Alkylation B) is a protein found in E. coli, induced during an adaptive response and involved in the direct reversal of alkylation damage.Errol C.Friedberg, Graham c. Walker, Wolfram Siede, Richard D. Wood, Roger A. schultz, Tom Ellenberge ...
enzyme is associated with the repair of damaged
DNA.
In plants, αKG-dependent dioxygenases are involved in diverse reactions in plant metabolism.
These include flavonoid biosynthesis,
and ethylene biosyntheses.
In mammals and humans, αKG-dependent dioxygenase have functional roles in biosyntheses (e.g. collagen biosynthesis
and L-carnitine biosynthesis
), post-translational modifications (e.g. protein hydroxylation), epigenetic regulations (e.g.
histone
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn a ...
and
DNA demethylation
), as well as sensors of
energy metabolism
Bioenergetics is a field in biochemistry and cell biology that concerns energy flow through living systems. This is an active area of biological research that includes the study of the transformation of energy in living organisms and the study of ...
.
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.
Catalytic mechanism
αKG-dependent dioxygenases catalyse oxidation reactions by incorporating a single oxygen atom from molecular oxygen (O
2) into their substrates. This conversion is coupled with the oxidation of the cosubstrate
αKG into succinate and carbon dioxide.
With labeled O
2 as substrate, the one label appears in the succinate and one in the hydroxylated substrate:
:R
3CH +
O2 +
−O
2CC(O)CH
2CH
2CO
2− → R
3C
OH + CO
2 +
−O
OCCH
2CH
2CO
2−
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
2 and forming an
Fe(IV)-oxo intermediate. This Fe=O center then oxygenates the substrate by an
oxygen rebound mechanism
In biochemistry, the oxygen rebound mechanism is the pathway for hydroxylation of organic compounds by iron-containing oxygenases. Many enzymes effect the hydroxylation of hydrocarbons as a means for biosynthesis, detoxification, gene regulatio ...
.
Alternative mechanisms have failed to gain support.
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.
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 N
2O triad binds to one face of the Fe center, leaving three labile sites available on the octahedron for binding αKG and O
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.
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),
a αKG-dependent dioxygenase that is involved in oxygen sensing,
and
isopenicillin N synthase
Isopenicillin N synthase (IPNS) is a non-heme iron protein belongig to the 2-oxoglutarate (2OG)-dependent dioxygenases oxidoreductase family. This enzyme catalyzes the formation of isopenicillin N from δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valin ...
(IPNS), a microbial αKG-dependent dioxygenase.
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 is the organic compound with the formula HO2CC(O)NHCH2CO2H. This colourless solid is used as an inhibitor of α-ketoglutarate-dependent enzymes.Hausinger, R. P."Fe(II)/α-ketoglutarate-dependent hydroxylases and related enzyme ...
(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).
Although they are potent inhibitors of αKG-dependent dioxygenase, they lack selectivity and hence sometimes being referred to as so-called 'broad spectrum' inhibitors.
Inhibitors that compete against the substrate were also developed, such as peptidyl-based inhibitors that target human
prolyl hydroxylase domain 2 (PHD2)
and
Mildronate
Meldonium (INN; trade name Mildronate, among others) is a limited-market pharmaceutical, developed in 1970 by Ivars Kalviņš at the USSR Latvia Institute of Organic Synthesis, and now manufactured by the Latvian pharmaceutical company Gri ...
, a drug molecule that is commonly used in Russia and Eastern Europe that target
gamma-butyrobetaine dioxygenase.
Finally, as αKG-dependent dioxygenases require molecular oxygen as a co-substrate, it has also been shown that gaseous molecules such as carbon monoxide
and nitric oxide
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.
For example, assays were developed to study ligand binding,
enzyme kinetics,
modes of inhibition
as well as protein conformational change.
Mass spectrometry is also widely applied. It can be used to characterise enzyme kinetics,
to guide enzyme inhibitor development,
study ligand and metal binding
as well as analyse protein conformational change.
Assays using spectrophotometry were also used,
for example those that measure 2OG oxidation,
co-product succinate formation
or product formation.
Other biophysical techniques including (but not limited to) isothermal titration calorimetry (ITC)
and electron paramagnetic resonance (EPR) were also applied.
Radioactive assays that uses
14C labelled substrates were also developed and used.
Given αKG-dependent dioxygenases require oxygen for their catalytic activity, oxygen consumption assay was also applied.
Further reading
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References
{{Reflist
Enzymes
Human 2OG oxygenases