Molecular Playground/Prolyl Hydroxylase Domain (PHD) Enzyme: Difference between revisions
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=== Structure === | === Structure === | ||
---- | ---- | ||
[[Image:PHD2.png|300 px|thumb|Fig. 1: Substrate bound (purple) and unbound (green) PHD2. Figure created with PyMol. PDB IDs 2G19, 3HQR.]] | |||
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 <scene name='Sandbox_Prolyl_Hydroxylase_Domain_(PHD)_Enzyme/Jelly_roll_fold/3'>(DSBH) fold motif</scene> 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.<ref name="review" /><ref>Schofield, C.J., Ratcliffe, P.J. "Signalling Bypoxia by HIF Hydroxylases." Biochemical and Biophysical Research Communications, August 24, 2005, 338, 617-626. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16139242 16139242]</ref><ref name="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 <scene name='Sandbox_Prolyl_Hydroxylase_Domain_(PHD)_Enzyme/Jelly_roll_fold/3'>(DSBH) fold motif</scene> 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.<ref name="review" /><ref>Schofield, C.J., Ratcliffe, P.J. "Signalling Bypoxia by HIF Hydroxylases." Biochemical and Biophysical Research Communications, August 24, 2005, 338, 617-626. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16139242 16139242]</ref><ref name="structure" /> | ||
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The <scene name='Sandbox_Prolyl_Hydroxylase_Domain_(PHD)_Enzyme/Active_site/2'>active site</scene>, 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 <scene name='Sandbox_Prolyl_Hydroxylase_Domain_(PHD)_Enzyme/Fe_binding_triad_sequence/2'>triad</scene>, 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.<ref name="structure" /> | The <scene name='Sandbox_Prolyl_Hydroxylase_Domain_(PHD)_Enzyme/Active_site/2'>active site</scene>, 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 <scene name='Sandbox_Prolyl_Hydroxylase_Domain_(PHD)_Enzyme/Fe_binding_triad_sequence/2'>triad</scene>, 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.<ref name="structure" /> | ||
Upon binding of substrate β2β3 loop region of PHD2 goes to a large conformational change. β2β3 loop moves toward the active site and closes the active site entrance and it is believed to be essential for catalytic function of PHD2. β2β3 loop region is mostly conserved in PHDs with some differences. Mutational studies on β2β3 loop region has showed that this loop is significant for substrate recognition of PHDs<ref>PMID:18063574</ref>. | Upon binding of substrate β2β3 loop region of PHD2 goes to a large conformational change (Figure 1). β2β3 loop moves toward the active site and closes the active site entrance and it is believed to be essential for catalytic function of PHD2. β2β3 loop region is mostly conserved in PHDs with some differences. Mutational studies on β2β3 loop region has showed that this loop is significant for substrate recognition of PHDs<ref>PMID:18063574</ref>. | ||
=== Function === | === Function === | ||
Revision as of 23:43, 12 December 2012
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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, PHD1 is found in the nucleus, PHD2 is mostly located in the cytoplasm and PHD3 is distributed in both [3] 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]. PHDs differ in terms of the proline hydroxylation, PHD2 hydroxylates Pro-402 and Pro-564 residue in oxygen degradation domain of (HIF)-α whereas PHD3 can only hydroxylate Pro-564 position.
Molecular Playground banner: Prolyl Hydroxylase Domain (PHD) enzyme, a cellular oxygen sensor, has a major regulatory role in oxygen homeostasis.
StructureStructure
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][4][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]
Upon binding of substrate β2β3 loop region of PHD2 goes to a large conformational change (Figure 1). β2β3 loop moves toward the active site and closes the active site entrance and it is believed to be essential for catalytic function of PHD2. β2β3 loop region is mostly conserved in PHDs with some differences. Mutational studies on β2β3 loop region has showed that this loop is significant for substrate recognition of PHDs[5].
FunctionFunction
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 in the oxygen degradation domain at position 4 of the residues Pro-402 (NODD) and Pro-564 (CODD) of hypoxia-inducible transcription factor, (HIF)-α.[1] Proline hydroxylation in NODD and CODD, enable recognition by the von Hippel Lindau (pVHL) ubiquitin ligase complex and flowing degradation by the proteasome[6] [7].
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, which 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[8]; 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
3D structures of hypoxia-inducible factor prolyl hydroxylase3D structures of hypoxia-inducible factor prolyl hydroxylase
Hypoxia-inducible factor prolyl hydroxylase
ReferencesReferences
- ↑ 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
- ↑ 2.0 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
- ↑ Metzen E, Berchner-Pfannschmidt U, Stengel P, Marxsen JH, Stolze I, Klinger M, Huang WQ, Wotzlaw C, Hellwig-Burgel T, Jelkmann W, Acker H, Fandrey J. Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing. J Cell Sci. 2003 Apr 1;116(Pt 7):1319-26. PMID:12615973
- ↑ Schofield, C.J., Ratcliffe, P.J. "Signalling Bypoxia by HIF Hydroxylases." Biochemical and Biophysical Research Communications, August 24, 2005, 338, 617-626. PMID:16139242
- ↑ Flashman E, Bagg EA, Chowdhury R, Mecinovic J, Loenarz C, McDonough MA, Hewitson KS, Schofield CJ. Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases. J Biol Chem. 2008 Feb 15;283(7):3808-15. Epub 2007 Dec 5. PMID:18063574 doi:10.1074/jbc.M707411200
- ↑ Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, Kaelin WG Jr. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 2001 Apr 20;292(5516):464-8. Epub 2001 Apr 5. PMID:11292862 doi:10.1126/science.1059817
- ↑ Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, von Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001 Apr 20;292(5516):468-72. Epub 2001 Apr 5. PMID:11292861 doi:10.1126/science.1059796
- ↑ Kaelin, W.G. "Proline Hydroxylation and Gene Expression." Annu.Rev.Biochem., February 8, 2005, 74, 115-128. PMID:15952883