Journal:JMB:3

<scene name='Journal:JMB:3/Cv/6'>Metal binding sites</scene>, especially those playing a catalytic role, exhibit high structural conservation (wildtype serum paraoxonase-1 (PON1) in the presence of either phosphate (PDB: [[3sre]]) or the lactone-­analogue 2HQ (PDB: [[3srg]])). The location of the metal ion and of its ligating residues perfectly superpose, even in distant superfamily members that catalyze different chemical reactions. There exist, however, indications of changes in the configuration of catalytic metals, as part of the catalytic cycle, or upon binding different substrates. Such example is the case of the serum paraoxonase-1 (PON1), in which mutations in the H115 active site residue, induce a <scene name='Journal:JMB:3/Cv/7'>shift towards an alternative coordination mode of the catalytic Ca2+</scene> (<span style="color:royalblue;background-color:black;font-weight:bold;">wildtype PON1 is in royalblue</span>, <font color='magenta'><b>H115W mutant is in magenta</b></font> and <span style="color:salmon;background-color:black;font-weight:bold;">H115Q/H134Q mutant is in salmon</span>). PON1's native activity is the hydrolysis of lipophilic lactones, but it also promiscuously hydrolyzes organophosphates (OPs), particularly paraoxon. It uses different subsets of its catalytic machinery, and different active-site conformations, to catalyze these two reactions. However, the catalytic Ca2+, and its ligating residues are essential for both. <scene name='Journal:JMB:3/Cv/9'>H115</scene> is playing a key role in the hydrolysis of lactones. Together with E53, it activates the hydrolytic water for the lactonase activity. Further, mutations to <scene name='Journal:JMB:3/Cv/10'>Gln</scene> or <scene name='Journal:JMB:3/Cv/11'>more drastically to Trp</scene>, reduce the lactonase activity significantly (up to 600-fold). The OP hydrolase activity is, however, enhanced. To gain insight for the structural and mechanistic changes that responsible for this functional transition, the crystal structure of two H115 mutants was determined, <scene name='Journal:JMB:3/Cv/14'>H115W</scene> and <scene name='Journal:JMB:3/Cv/13'>H115Q/H134Q</scene>. These crystal structures display major rearrangements of the catalytic metal and of its ligating residues. Specifically, the <scene name='Journal:JMB:3/Cv/7'>catalytic Ca2+ has moved</scene> a 1.8 Å upwards towards the enzyme’s surface, relative to its position in the WT structure. The position of the <scene name='Journal:JMB:3/Cv/15'>structural Ca2+, however, did not change</scene>. Further, the residues coordinating the catalytic Ca2+ are also altered in the mutant structure- the side-chains of <scene name='Journal:JMB:3/Cv/16'>N168, N224 and N270 do not interact directly with the catalytic Ca2+ as in the WT structure, but through waters that are absent in the native structure</scene>. The side-chain of <scene name='Journal:JMB:3/Cv/17'>N224 also exhibits a different orientation</scene>. For <scene name='Journal:JMB:3/Cv/18'>E53, which retains its direct interaction with the catalytic Ca2+, an alternative side-chain conformation was detected</scene>. Finally, the side-chains in the vicinity of the mutations also moved, for example for the <scene name='Journal:JMB:3/Cv/19'>H115W mutant, residues H134 and L69 moved</scene> in order to accommodate the bulkiness of the Trp in position 115. The structural strudies were also complimented with biochemical, mutational and computational analysis that were in good agreement with the structural observations. The computational simulations also suggest a general base catalysis mechanism in which <scene name='Journal:JMB:3/Cv/24'>E53, possibly together with H115 and/or D269</scene>, coordinates and activates the attacking water molecule. These findings, taken together, support the notion that PON1 can accommodate <scene name='Journal:JMB:3/Cv/7'>two (or more) alternative coordination modes for its catalytic Ca2+</scene>, and that these modes may be used to catalyze different reactions. PON1's native lactonase activity occurs within the <scene name='Journal:JMB:3/Cv/21'>canonical coordination scheme</scene>, with the location of the catalytic Ca2+ being similar in PON1 and in related enzymes that are highly diverged in their sequences. The promiscuous OPH activity, however, seems to utilize a <scene name='Journal:JMB:3/Cv/23'>fundamentally different Ca2+ mode</scene>, and a different mechanism. Alongside the conformational diversity of the protein's backbone and side-chains, metal repositioning may, therefore, contribute to the catalytic versatility of enzymes and to the ease by which new enzymatic functions diverge. The shift in the Ca2+ position, from a rarely populated metal state in the WT to a dominant state in H115W, follows a general model whereby evolution capitalizes on stochastic variations, be they atomic as with PON1's alternative location of the Ca2+, or cellular (''e.g.'', transcriptional noise). Mutations do not create something from nothing. Rather, they shift the distribution such that a marginal, noise phenomenon becomes the norm.

<scene name='Journal:JMB:3/Cv/6'>Metal binding sites</scene>, especially those playing a catalytic role, exhibit high structural conservation (wildtype serum paraoxonase-1 (PON1) in the presence of either phosphate (PDB: [[3sre]]) or the lactone-­analogue 2HQ (PDB: [[3srg]])). The location of the metal ion and of its ligating residues perfectly superpose, even in distant superfamily members that catalyze different chemical reactions. There exist, however, indications of changes in the configuration of catalytic metals, as part of the catalytic cycle, or upon binding different substrates. Such example is the case of the serum paraoxonase-1 (PON1), in which mutations in the H115 active site residue, induce a <scene name='Journal:JMB:3/Cv/7'>shift towards an alternative coordination mode of the catalytic Ca2+</scene> (<span style="color:royalblue;background-color:black;font-weight:bold;">wildtype PON1 is in royalblue</span>, <font color='magenta'><b>H115W mutant is in magenta</b></font> and <span style="color:salmon;background-color:black;font-weight:bold;">H115Q/H134Q mutant is in salmon</span>). PON1's native activity is the hydrolysis of lipophilic lactones, but it also promiscuously hydrolyzes organophosphates (OPs), particularly paraoxon. It uses different subsets of its catalytic machinery, and different active-site conformations, to catalyze these two reactions. However, the catalytic Ca2+, and its ligating residues are essential for both. <scene name='Journal:JMB:3/Cv/9'>H115</scene> is playing a key role in the hydrolysis of lactones. Together with E53, it activates the hydrolytic water for the lactonase activity. Further, mutations to <scene name='Journal:JMB:3/Cv/10'>Gln</scene> or <scene name='Journal:JMB:3/Cv/11'>more drastically to Trp</scene>, reduce the lactonase activity significantly (up to 600-fold). The OP hydrolase activity is, however, enhanced. To gain insight for the structural and mechanistic changes that responsible for this functional transition, the crystal structure of two H115 mutants was determined, <scene name='Journal:JMB:3/Cv/14'>H115W</scene> and <scene name='Journal:JMB:3/Cv/13'>H115Q/H134Q</scene>. These crystal structures display major rearrangements of the catalytic metal and of its ligating residues. Specifically, the <scene name='Journal:JMB:3/Cv/7'>catalytic Ca2+ has moved</scene> a 1.8 Å upwards towards the enzyme’s surface, relative to its position in the WT structure. The position of the <scene name='Journal:JMB:3/Cv/15'>structural Ca2+, however, did not change</scene>. Further, the residues coordinating the catalytic Ca2+ are also altered in the mutant structure- the side-chains of <scene name='Journal:JMB:3/Cv/16'>N168, N224 and N270 do not interact directly with the catalytic Ca2+ as in the WT structure, but through waters that are absent in the native structure</scene>. The side-chain of <scene name='Journal:JMB:3/Cv/17'>N224 also exhibits a different orientation</scene>. For <scene name='Journal:JMB:3/Cv/18'>E53, which retains its direct interaction with the catalytic Ca2+, an alternative side-chain conformation was detected</scene>. Finally, the side-chains in the vicinity of the mutations also moved, for example for the <scene name='Journal:JMB:3/Cv/19'>H115W mutant, residues H134 and L69 moved</scene> in order to accommodate the bulkiness of the Trp in position 115. The structural strudies were also complimented with biochemical, mutational and computational analysis that were in good agreement with the structural observations. The computational simulations also suggest a general base catalysis mechanism in which <scene name='Journal:JMB:3/Cv/24'>E53, possibly together with H115 and/or D269</scene>, coordinates and activates the attacking water molecule. These findings, taken together, support the notion that PON1 can accommodate <scene name='Journal:JMB:3/Cv/7'>two (or more) alternative coordination modes for its catalytic Ca2+</scene>, and that these modes may be used to catalyze different reactions. PON1's native lactonase activity occurs within the <scene name='Journal:JMB:3/Cv/21'>canonical coordination scheme</scene>, with the location of the catalytic Ca2+ being similar in PON1 and in related enzymes that are highly diverged in their sequences. The promiscuous OPH activity, however, seems to utilize a <scene name='Journal:JMB:3/Cv/23'>fundamentally different Ca2+ mode</scene>, and a different mechanism. Alongside the conformational diversity of the protein's backbone and side-chains, metal repositioning may, therefore, contribute to the catalytic versatility of enzymes and to the ease by which new enzymatic functions diverge. The shift in the Ca2+ position, from a rarely populated metal state in the WT to a dominant state in H115W, follows a general model whereby evolution capitalizes on stochastic variations, be they atomic as with PON1's alternative location of the Ca2+, or cellular (''e.g.'', transcriptional noise). Mutations do not create something from nothing. Rather, they shift the distribution such that a marginal, noise phenomenon becomes the norm.

</StructureSection>

</StructureSection>

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