Journal:JMB:2

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<applet load="1stp" size="500" color="" frame="true" spin="on" Scene ="" align="right" caption=" caption ''"/>
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<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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=== Title Of The Paper ===
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=== Catalytic versatility and backups in enzyme active sites: The case of serum paraoxanase 1 ===
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<big>Authors</big><ref >none yet</ref>
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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>
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<b>Molecular Tour</b><br>
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==Molecular Tour==
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The source of diversity in life is a parallel diversity in the atoms of DNA - add a few atoms here, take some away there, and behold! the lion had become a lamb. Proteins, the main building stuff of life, are the primary recipients of changes in DNA atmos: fiddle with DNA, increase the diversity of proteins, of life. But how does an old protein morph into a new one? How does a protein that is comprised of thousands of atoms, change as a result of changes in a few DNA atoms?
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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.
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Farmers in the 1960s were using a man-made pesticide called “parathion” that interferred with insects’ nervous systems. But the 1980s, scientists had found that some bacteria on these farms contained an paraoxanse enzyme, they called Phosphotriesterase (PTE) for eating this pesticide. How did the bacteria evolve this enzyme in just a few decade? The recent paper by Ben David et al. (JMB, 2012) of the Weizmann Institute offers a compelling explanation for the evolution of paraoxon-metabolizing enzymes in a short period, and their explanation begins to explain how the tweaking of atoms in DNA has resulted in the glorious diversity of life.
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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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The discovery several years ago that a lactonase enzyme they called Quarom quenching lactonase (QQL) was the parent of the novel PTE, as demonstrated by similar fold, active site configuration, and sequence, was an exciting first step. The lab had since demonstrated that a lactonase from mammals (PON1) (shown - helix, beta sheet and active site) can be turned into a paraoxanase by changing just one amino amino acid (shown) By analogy, understanding how PON1 becomes a paraoxonase will likely explain the natural evolution of QQL to PTE.
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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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PON1 uses more than one amino acid (shown - H115 and E53) to achieve a single function (serving as bases for activating a water to become a nucleophile), and this the researchers think is key to PON1 becoming a paraoxanse. Lactone, the intended substrate of PON1, is the right shape to make use of both H115 and E53 (shown), but paraoxon has a similar but not identical shape, and can only take advantage of E53 (shown). But because the bacteria has an enzyme with already some paraoxon activity, it only needs to tweak the protein to achieve higher paraoxon activity, and tweaking DNA can accomplish just that. The use of two amino acids to do one function enables similar but not identical molecules to use even one amino acid to achieve that function. One amino acid at least gives paraoxon a “hand-hold” on the protein that facilitates further evolution. The key to evolution is additive distribution of one function between amino acids. Thus, a non-native molecule can at least make use of one amino acid. And natural selection takes over from there.
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Importantly, in a world fearful of chemical warfare, Paroxonase can be used to prevent nerve agents like paroxon from mortally damaging a person’s nervous system.
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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]].
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</StructureSection>
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Current revision

PON1 - looking down 6-bladed propellers, Ca+2 and 2HQ

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  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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