Journal:JMB:2

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<big>Moshe Ben-David, Mikael Elias, Jean-Jacques Filippi, Elisabet Dunach, Israel Silman, Joel Sussman and Dan Tawfik, PhD</big> <ref >doi 10.1016/j.jmb.2012.02.042</ref>
<big>Moshe Ben-David, Mikael Elias, Jean-Jacques Filippi, Elisabet Dunach, Israel Silman, Joel Sussman and Dan Tawfik, PhD</big> <ref >doi 10.1016/j.jmb.2012.02.042</ref>
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==Overview==
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==Molecular Tour==
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Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate detoxification and in prevention of atherosclerosis. There are three members in this family, PON1, PON2 and PON3, which share 60-70% nucleic acid identity. The most studied enzymes are the two isoenzymes of PON1, which differ in the residue at position 192 (Q/R). The primary activity of PON1 is lactone hydrolysis; however, this enzyme has many other activities. One of the interesting activities is the hydrolysis of organophosphates. PON1 can catalyze a variety of nerve agents such as cyclosarin, soman, etc. Therefore, it is aimed to be a nerve agent scavenger. In addition, PON1 has a role in prevention of atherosclerosis and is found to be attached to the high-density lipoprotein (HDL, “good cholesterol”).
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<scene name='Journal:JMB:2/Scene_1_2/1'>TextToBeDisplayed</scene>
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Human PON1 is not stable, and tends to aggregate in the absence of detergents. In addition, it cannot be expressed in bacteria or yeast for protein over-expression, mutagenesis and protein engineering. Therefore, this protein was submitted to directed evolution in order to over-express it in ''E.coli'' and to increase its solubility. Family shuffling of four PON1 genes (human, mouse, rabbit and rat) resulted in many variants that could be expressed in ''E.coli'', but only one of them (G2E6-variant) led to quality-diffracted crystals. The recombinant-PON1 (rePON1) G2E6 variant, exhibits 91% homology to the wt rabbit PON1 and 86% homology to the human PON1. This variant exhibits resemble catalytic activity to human PON1.
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==Structural features==
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The crystal structure of rePON1 shows<ref>PMID: 15098021 </ref><ref>PMID: 21217689 </ref> a <scene name='1v04/Six_blad/8'>six-bladed</scene> β-propeller fold. PON1 has a unique addition to the β-propeller scaffold: three <scene name='1v04/Three_helixes_pon1/9'> α-helixes</scene>, which are located on the top of the propeller. These helixes are likely to be involved in the anchoring to the [[HDL]] particles. In addition, a stabilizing <scene name='1v04/Cys_bridge/13'>disulfide bridge</scene> between Cys-42 and Cys-353 was found. The structure of rePON1 resembles that of <scene name='1v04/Loligo_v_structure/4'>''Loligo vulgaris'' DFPase</scene> (PDB [[1e1a]]). Both are six-bladed propellers with each blade consisting of four β-sheets. Moreover, in both structures two <scene name='1v04/Two_ca_ions/5'>calcium ions</scene> can be found in their central tunnel. The calcium atom, which resides at the top of the tunnel, is assigned as the ‘catalytic calcium’ (Ca-1), whereas the other calcium at the central section is assigned as the ‘structural calcium’ (Ca-2). The latter is involved in stabilization of the structure. In addition to the two calciums, there is a <scene name='1v04/Po4_ion/7'>phosphate ion</scene> , which is bound to Ca-1 in the active site. This phosphate ion is thought to come from the mother liquor. The Loligo vulgaris structure lacks the three α-helixes found in the rePON1 structure.
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The catalysis mechanism of organophosphates has yet not been discovered. However, determination of the pH-rate profile of rePON1 proposed participation of a Histidine (His) dyad in the [[lactonase]] activity of PON1. In hydrolytic enzymes, His often serves as a base, deprotonating a water molecule, and thus generating the attacking hydroxide ion that produces hydrolysis. The <scene name='1v04/His_dyad/22'>His-dyad</scene> (His-115 and His-134) resides near both Ca-1 and the phosphate ion. The hypothesis is that His-115 acts as a general base to deprotonate a single water molecule, thus generating the attacking hydroxide, while His-134 acts in a proton shuttle mechanism to increase His-115’s basicity. In addition, His-115 was found to have distorted dihedral angles, thing that characterizes many catalytic residues. This observation was supported by site mutation of both His-115 and His-134, which result in a dramatic decrease in both arylesterase and lactonase activity of PON1. Interestingly, the organophosphate hydrolysis activity of these mutations was not affected. Therefore, different loactions in the rePON1 <scene name='1v04/Pon1_activesite/5'>binding site</scene> are postulated to have different enzymatic activities in its
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<scene name='1v04/Rasmol_tst_01/1'> overall structure</scene>.
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Previously PON1 was <scene name='Journal:JMB:2/Scene_2/2'>solved at 4.5 pH</scene>. We sought a physiologically active pH and <scene name='Journal:JMB:2/Scene_2/3'>solved PON1 at 6.5 pH (overlain with 4.5)</scene>. Note <scene name='Journal:JMB:2/Scene_3/2'>residues 346-348 in the two structures</scene>. Especially, observe the <scene name='Journal:JMB:2/Scene_4/1'>movement of residue 71</scene>. We also solved PON1 at 6.5 pH in <scene name='Journal:JMB:2/Scence_5/3'>complex with 2HQ (a lactone approximate)</scene>. Here, we for the first time observe ordered <scene name='Journal:JMB:2/Scene_6/1'>active site loop density</scene>. The residues colored red <scene name='Journal:JMB:2/Scene_7/1'>contact the active site</scene>. <scene name='Journal:JMB:2/Scene_8/1'>2HQ overlaps with PO4</scene>, suggesting that lactone adopt a similar position. 2HQ makes contact with <scene name='Journal:JMB:2/Scene_9/1'>several catalytic residues</scene>.
Previously PON1 was <scene name='Journal:JMB:2/Scene_2/2'>solved at 4.5 pH</scene>. We sought a physiologically active pH and <scene name='Journal:JMB:2/Scene_2/3'>solved PON1 at 6.5 pH (overlain with 4.5)</scene>. Note <scene name='Journal:JMB:2/Scene_3/2'>residues 346-348 in the two structures</scene>. Especially, observe the <scene name='Journal:JMB:2/Scene_4/1'>movement of residue 71</scene>. We also solved PON1 at 6.5 pH in <scene name='Journal:JMB:2/Scence_5/3'>complex with 2HQ (a lactone approximate)</scene>. Here, we for the first time observe ordered <scene name='Journal:JMB:2/Scene_6/1'>active site loop density</scene>. The residues colored red <scene name='Journal:JMB:2/Scene_7/1'>contact the active site</scene>. <scene name='Journal:JMB:2/Scene_8/1'>2HQ overlaps with PO4</scene>, suggesting that lactone adopt a similar position. 2HQ makes contact with <scene name='Journal:JMB:2/Scene_9/1'>several catalytic residues</scene>.

Revision as of 19:23, 21 March 2012

PON1 - looking down 6-bladed propellers, Ca+2 and phosphate ions seen 1v04

Drag the structure with the mouse to rotate
  1. ↑ Ben-David M, Elias M, Filippi JJ, Dunach E, Silman I, Sussman JL, Tawfik DS. Catalytic Versatility and Backups in Enzyme Active Sites: The Case of Serum Paraoxonase 1. J Mol Biol. 2012 Mar 1. PMID:22387469 doi:10.1016/j.jmb.2012.02.042

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