Sandbox Prolyl Hydroxylase Domain (PHD) Enzyme

From Proteopedia

One of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground.

Metazoans adapt to oxygen levels in the environment by making use of intracellular oxygen levels as signals to regulate the transcription of genes that are essential under normoxic or hypoxic conditions. Central to this mechanism is the oxygen-dependent hydroxylation on specific proline and asparagine residues of the transcription factor, hypoxia-inducible factor (HIF)-α.^[1]

Prolyl hydroxylase domain (PHD) enzyme (EC 1.14.11.-) is a Fe(II)/2-oxoglutarate (OG)-dependent dioxygenase that catalyzes the trans-4-hydroxylation of the specific proline residues (in humans, Pro-402 and Pro-564) in (HIF)-α. In addition to iron, this enzyme also requires ascorbate as a cofactor.^[2]

PHDs belong to the same oxygenase superfamily as the collagen prolyl hydroxylases. Inside the cell, these proteins are mostly found in the cytoplasm in contrast to collagen prolyl hydroxylases, which reside in the endoplasmic reticulum. In mammals, the PHD dioxygenase subfamily originally includes three homolog members but was recently updated to include another member: PHD1 (also known as HPH3 and EGLN2), PHD2 (also known as HPH2 and EGLN1), PHD3 (also known as HPH1 and EGLN3), and a newly identified enzyme called P4H-TM (also recently named PHD4 and EGLN4). Both PHD1 and PHD2 contain more than 400 amino acid residues while PHD3 has less than 250. All isoforms, however, contain the highly conserved hydroxylase domain in the catalytic carboxy-terminal region. ^[1]

PDB ID 2g19 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
2g19, resolution 1.70Å ()
Ligands:	,
Structural annotation: Resources: InterPro : Ipr002893, Ipr005123, Ipr006620 Pfam : PF03171 UniProt : Q9GZT9
Functional annotation: Cellular component: cytosol Biological process: protein metabolic process Molecular function: protein binding oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen L-ascorbic acid binding metal ion binding oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors iron ion binding zinc ion binding oxidoreductase activity
Evolutionary conservation: Toggle Conservation Colors: Rows = identical sequences: Further Information: Caveats, Explanation, Complete Results at ConSurfDB
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

Structure

PHDs have two structural domains: the more variable N-terminal domain and the conserved catalytic C-terminal domain. The catalytic domain core of PHDs consists of eight β-strands in a "jelly-roll" or double stranded β helix supported by three conserved α-helices and other β-strands and loops that pack along the core. Possession of the DSBH motif is typical of 2-OG-dependent oxygenases. Contained in this core are the three Fe(II)-binding ligands formed by the conserved triad sequence, His-X-Asp/Glu-Xn-His.^[1][3][2]

The , which is located on a deep cleft between the β-strands comprising the DBSH core, contains the essential Fe(II). It is normally coordinated by the conserved two-histidine-one-carboxylate , 2-OG and a water molecule to form an octahedral geometry. Aside from the triad motif residues and those that bind 2-OG, the residues that are predominant inside the active site are nonpolar in nature. This is evidence of the enzyme's need to protect the protein core from oxidation by reactive species that are sometimes generated from iron-related reactions like the Fenton type reaction.^[2]

Function

The intrinsic dependence of PHD-catalyzed hydroxylation reactions on molecular oxygen concentration led to the most notable role of PHDs as cellular oxygen sensors. The hydroxylation happens at position 4 of the residues Pro-402 and Pro-564 located in the C-terminal oxygen-dependent degradation domains (ODDs) of the hypoxia-inducible transcription factor, (HIF)-α.^[1]

The requirement of PHDs for the TCA cycle intermediate, 2-oxoglutarate, also opens the possibility of these enzymes acting as regulators of processes that relate metabolic activity to oxygen levels. Aside from regulation of oxygen homeostasis, other biological functions of the enzyme, that may be hydroxylase-independent or still hydroxylase-dependent but (HIF)-α-independent, are being proposed. This is mainly based on the results of various studies: some showed that other factors such as nitric oxide, reactive oxygen species (ROS), and several oncogenes control PHD oxygenase activity^[4]; while others described PHD activity on other substrates like IKK-β^[1]. In fact, several functions of the enzyme have been recently identified based on these studies. Listed below are the currently identified functions for PHDs in general^[1]:

• tumor suppressor
• promoter of cell death (apoptosis)
• regulator of cell differentiation

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4 1.5} Fong, G.H., Takeda, K. "Role and Regulation of Prolyl Hydroxylase Domain Proteins." Cell Death and Differentiation, February 15, 2008, 15, 635-641. PMID:18259202
2. ↑ ^{2.0 2.1 2.2} Mcdonough, M.A., Li, V., Flashman, E., et al. "Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2)." PNAS, June 27, 2006, 103 (26), 9814-9819. PMID:16782814
3. ↑ Schofield, C.J., Ratcliffe, P.J. "Signalling Bypoxia by HIF Hydroxylases." Biochemical and Biophysical Research Communications, August 24, 2005, 338, 617-626. PMID:16139242
4. ↑ Kaelin, W.G. "Proline Hydroxylation and Gene Expression." Annu.Rev.Biochem., February 8, 2005, 74, 115-128. PMID:15952883