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| - | <StructureSection load='1v04' size='400' side='right' caption='PON1 - looking down 6-bladed propellers, Ca+2 and phosphate ions seen [[1v04]]' scene='1v04/Starting_scene/3'> | + | <StructureSection load='1v04' size='400' side='right' caption='PON1 - looking down 6-bladed propellers, Ca+2 and 2HQ' scene='Journal:JMB:2/Opening/4'> |
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| | === Catalytic versatility and backups in enzyme active sites: The case of serum paraoxanase 1 === | | === Catalytic versatility and backups in enzyme active sites: The case of serum paraoxanase 1 === |
| | <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> |
| | <hr/> | | <hr/> |
| - | <b>Molecular Tour</b><br>
| + | ==Molecular Tour== |
| - | | + | The PON1 enzyme has theoretical biological importance as well as application for treatment of neurotoxins. Questions of the origins of enzyme promiscuity or the evolution of protein diversity may be illuminated by PON1's accidental low-level phosphotriesterase activity, and by the unintuitive effect of switching one amino acid in PON1 whereby it changes from a lactonase to a phosphotriesterase. Practically, a potent neurotoxin, "Paraoxon", and therefore a biochemical warfare threat, can be neutralized by phosphotriesterases. |
| - | ==Overview==
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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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| - | 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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| | + | See also [[Serum Paraoxonase]] for a general review of issues relating to PON1 and the overall class of Serum Paraoxonases. A initial structural tour begins there as well. |
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| - | Previously PON1 was <scene name='Journal:JMB:2/Scene_1/3'>solved at 4.5 pH</scene>. We sought a physiologically active pH and <scene name='Journal:JMB:2/Scene_2/1'>solved PON1 at 6.5 pH (overlain with 4.5)</scene>. Note <scene name='Journal:JMB:2/Scene_3/1'>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>.
| + | We experimentally solved two critical new PON1 structures. Previously solved in <scene name='Journal:JMB:2/Scene_1_2/3'>non-physiological conditions of pH 4.5</scene>, we have solved PON1 in <scene name='Journal:JMB:2/Scene_2_2/2'>physiological conditions of pH 6.5</scene>. While <scene name='Journal:JMB:2/Scene_3_2/1'>generally similar</scene>, as expected, there are some key differences. The side-chain of V346 within the active site pocket is <scene name='Journal:JMB:2/Scene_4_2/1'>rotated relative to the pH 4.5 structure</scene>, and the side-chains of F347 and H348 in the active site's 'second shell' <scene name='Journal:JMB:2/Scene_5_2/1'>adopted completely different rotamers</scene>. |
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| - | <scene name='Journal:JMB:2/Scene_10/3'>Changes in the rePON1 binding site upon binding of 2HQ</scene> (<scene name='Journal:JMB:2/Scene_10/2'>without labels</scene>). Superimposition of the rePON1- 2HQ complex (cyan; the closed conformation) with the apo rePON1 structures at pH 4.5 (orange) and pH 6.5 (blue) (the open conformations). The pH 4.5 conformation prevents closure of the active- site loop due to clashes of F347 and H348 with the loop residues (e.g. F77 and I74). Also illustrated is the movement of Y71 (dashed arrow) upon binding of 2HQ, and its interaction with D183 in the 2HQ complex structure. In summary, there are | + | Next, we crystallized <scene name='Journal:JMB:2/Scene_6_2/2'>PON1 in complex with 2-hydroxyquinoline (2HQ)</scene>, which is a lactone analog. As expected, this structure was also <scene name='Journal:JMB:2/Scene_7_2/1'>generally similar to the one at pH 4.5</scene> and <scene name='Journal:JMB:2/Scene_8_2/1'>pH 6.5</scene>. We could now see an <scene name='Journal:JMB:2/Scene_7b_2/1'>active site loop, residues 71-81</scene>, most of which had not been seen at either pH 4.5 or 6.5. The first segment of the active site loop, and <scene name='Journal:JMB:2/Scene_8b_2/1'>residues Y71 and I74 in particular</scene>,comprises part of PON1's active-site wall. Further, 2HQ's carbonyl oxygen and NH moiety <scene name='Journal:JMB:2/Scene_9_2/1'>overlap with the phosphate oxygens</scene> in the apo structure. This overlap supports the notion that both the phosphate ion and 2HQ mimic the binding mode of substrates and/or reaction intermediates. In addition to interacting with the catalytic calcium, 2HQ interacts with the<scene name='Journal:JMB:2/Scene_10_2/1'> side-chains of H115, D269, E53 and N168</scene>. Importantly, while the bound 2HQ is in contact with the <scene name='Journal:JMB:2/Scene_11_2/1'>side-chains of Y71</scene>, in the absence of ligand Y71 is either disordered (pH 6.5), or (pH 4.5) <scene name='Journal:JMB:2/Scene_12_2/1'>positioned outside the binding pocket </scene>. |
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| | + | '''PDB references:''' Serum paraoxonase-1 by directed evolution at pH 6.5, [[3sre]]; Serum paraoxonase-1 by directed evolution at pH 6.5 in complex with 2-hydroxyquinoline, [[3srg]]. |
| | </StructureSection> | | </StructureSection> |
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| | <references/> | | <references/> |
| | __NOEDITSECTION__ | | __NOEDITSECTION__ |
