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| =='''Isocitrate Lyase from ''Mycobacterium tuberculosis'''''== | | {{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> |
| <StructureSection load='1F8I' size='340' side='right' caption='Isocitrate Lyase' scene='> | | ==Your Protein Name here== |
| ==Introduction==
| | <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''> |
| [http://www.rcsb.org/pdb/explore/explore.do?structureId=1f8i Isocitrate lyase] is a [[lyase]] found in the proteome of multiple bacteria that oxidizes the hydroxl group of [https://en.wikipedia.org/wiki/Isocitric_acid isocitrate] and cleaves the substrate in two forming [https://en.wikipedia.org/wiki/Glyoxylic_acid glyoxylate] and [https://en.wikipedia.org/wiki/Succinic_acid succinate]. Isocitrate lyase is a tetramer that is composed primarily of alpha helices and beta sheets with a unique structural phenomenon called "<scene name='69/694225/Helix_swapping/1'>helix swapping</scene>". This enzyme can be found within the cytosol of bacteria and is used in a variation of the citric acid cycle to help conserve energy by not using [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide_phosphate NADPH] as an electron carrier and by reforming [http://en.wikipedia.org/wiki/Coenzyme_A coenzyme-A] earlier than in the normal citric acid cycle.
| | This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs. |
| ==Isocitrate Lyase==
| | You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue. |
| ===Structure===
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| [[Image:Normal_Crystal_Structure.png|250 px|left|thumb|'''Figure 1. Crystal Structure of Isocitrate Lyase.''' Quaternary structure is comprised of four subunits forming an α/β barrel. A side view is shown where each comprising subunit is a different color with the central hole of the barrel coming perpendicularly out of the page.]]
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| <scene name='69/694225/Isocitrate_lyase/4'>Isocitrate lyase</scene> is a tetramer with 222 symmetry. Each <scene name='69/694225/Subunit_a/2'>subunit</scene> is composed of 14 alpha helices and 14 beta sheets which includes a total of 426 residues. These α helices and β sheets form an unusual <scene name='69/694225/Beta_barrel/1'>α/β barrel</scene>. The α/β barrel contains a topology of (βα)<sub>2</sub>α(βα)<sub>5</sub>β, differing from the canonical (βα)<sub>8</sub> pattern. Residues 184-200 and 235-254 connects the third and forth β-strands to their consecutive helices and form a <scene name='69/694225/Beta_domain/1'>small β-domain</scene> that consists of a short five-stranded βsheet (β6,β7,β9,β10,β11) that lies on top of the α/β barrel. <ref name="sharma"> Sharma, V.; Sharma, S.; Hoener zu Bentrup, K.; McKinney, J.; Russell, D.; ''et. al''; Structure of isocitrate lyase, a persistence factor of ''Mycobacterium tuberculosis''. ''Nat. Struct. Biol.''. '''2000'''. ''7(8)'':663-668. </ref> Additionally, this β-domain contains the catalytic loop necessary for isocitrate lyase to breakdown isocitrate. A study of the equilibria between the <scene name='69/694225/Isocitrate_lyase/4'>four subunits</scene> shows that each isocitrate lyase monomer has a dynamic comformational change of the active site loop. At any given time, only two of the subunits are in the open conformation. <ref name="gould"> Gould, T.; van de Langemheen, H.; Muñoz-Elías, E.; McKinney, D.; Sacchettini, J.; Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in ''Mycobacterium tuberculosis''. ''Molecular Microbiology''. '''2006'''. ''61(4)'':940-947. doi:10.1111/j.1365-2958.2006.05297.x. </ref> Furthermore, isocitrate lyase shows a resemblance to [http://www.rcsb.org/pdb/explore/explore.do?structureId=1S2V phosphoenolpyrvate mutase]. <ref name="sharma"> Sharma, V.; Sharma, S.; Hoener zu Bentrup, K.; McKinney, J.; Russell, D.; ''et. al''; Structure of isocitrate lyase, a persistence factor of ''Mycobacterium tuberculosis''. ''Nat. Struct. Biol.''. '''2000'''. ''7(8)'':663-668. </ref>
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| | == Biological Function == |
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| ===Helix Swapping=== | | == Structural Overview == |
| A unique structural feature of this enzyme is a phenomenon called "<scene name='69/694225/Helix_swapping/1'>helix swapping</scene>".
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| Helix swapping is observed between two monomers to form stable dimers. The 12th and 13th helices of each monomer exchange three dimensional placement with the respective helices of the opposite monomer. Due to the 222 symmetry observed, only two dimers are present in the quaternary structure that then combine to form the observed tetramer. As a result of this structure, 18% of the surface of each monomer is buried within the protein.
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| | == Mechanism of Action == |
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| ===Active Site=== | | == Zinc Ligand(s) == |
| [[Image:Active Site Residues.png|250 px|left|thumb|'''Figure 2. Active Site Residues.''' All eight active site residues necessary for catalysis of isocitrate are shown in slate. However, the protein shown is a C191S mutant of isocitrate lyase.]] [[Image:Active_Site_Hydrogen_Bonding.png|250 px|right|thumb|'''Figure 3. Active site residues hydrogen bound to a cofactor and the products of the catalyzed isocitrate reaction.''' Glyoxylate is shown in blue, succinate is shown in green, and the Mg<sup>2+</sup> cofactor is shown in yellow.]] The active site of isocitrate lyase consists of eight residues: Trp93, Cys191, His193, Ser315, Ser317, Asn313, Thr347, Leu348 ('''Figure 2'''). Additionally, there are several other amino acid side chains present that form hydrogen bonding opportunities with isocitrate to catalyze the breakdown to glyoxylate and succinate. Ser91, Trp93, and Arg228 (all in green) form hydrogen bonds with <scene name='69/694225/Glyoxylate_hydrogen_bonding/6'> glyoxylate </scene> (pink). Mg<sup>2+</sup> (cyan) is also shown as a reference. The Asn313, Arg228, and Gly192 residues (all in green) <scene name='69/694224/Succinate_hydrogen_bonding/3'>hydrogen bond </scene> to one carboxylate within succinate and while the Ser315, Ser317, and His193 residues (all in cyan) form hydrogen bonds with the other carboxylate within succinate. <ref name="sharma"> Sharma, V.; Sharma, S.; Hoener zu Bentrup, K.; McKinney, J.; Russell, D.; ''et. al''; Structure of isocitrate lyase, a persistence factor of ''Mycobacterium tuberculosis''. ''Nat. Struct. Biol.''. '''2000'''. ''7(8)'':663-668. </ref> Additionally, a Mg<sup>2+</sup> ion is needed for further electrostatic stabilization of the extreme negative charge on isocitrate. This Mg<sup>2+</sup> hydrogen bonds to the carboxylate in glyoxylate and one of the carboxylates in succinate ('''Figure 3''').
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| | == Other Ligands == |
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| | | This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. |
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| ===Catalytic Loop===
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| [[Image:Active Loop Shift.png|250 px|left|thumb|'''Figure 4. Active Site Loop Shift.''' Binding of the ligand to the enzyme results in a conformational shift that facilitates the breakdown of isocitrate. The active site loop without a ligand bound is shown in wheat while the active site loop with a ligand bound is shown in green. The ligands are shown in raspberry.]] The <scene name='69/694225/Normal_catalytic_loop/4'>catalytic loop</scene> of isocitrate lyase consists of residues 185-196 ('''Figure 4'''). The two most important residues within the loop are <scene name='69/694225/Normal_catalytic_loop/3'>Cys191 and His193</scene> as these form a charge relay strong enough to extract a proton from isocitrate. Poor electron density has been observed for residues His193 and Leu194 indicating that this loop is very flexible. <ref name="sharma"> Sharma, V.; Sharma, S.; Hoener zu Bentrup, K.; McKinney, J.; Russell, D.; ''et. al''; Structure of isocitrate lyase, a persistence factor of ''Mycobacterium tuberculosis''. ''Nat. Struct. Biol.''. '''2000'''. ''7(8)'':663-668. </ref> This data backs up the claim that that monomers of the protein are in a structural equilibria between the open and closed forms of the active site. In order for the catalytic loop to shift into the closed position necessary for catalysis, isocitrate must be within the binding pocket. The hydrogen bonding opportunities formed cause a ripple effect that shifts the catalytic loop into a closer position. <ref name="sharma"> Sharma, V.; Sharma, S.; Hoener zu Bentrup, K.; McKinney, J.; Russell, D.; ''et. al''; Structure of isocitrate lyase, a persistence factor of ''Mycobacterium tuberculosis''. ''Nat. Struct. Biol.''. '''2000'''. ''7(8)'':663-668. </ref> This shift also causes the C-terminal domain (cyan) of the subunit (residues 411-428) to <scene name='69/694225/C-terminus_loop_in_cat_loop/3'>move</scene> into the former position of the catalytic loop (green). Also shown as a reference is the ligand (pink). The C-terminal domain is then stabilized by an <scene name='69/694225/Lys_electrostatic/4'>electrostatic interaction</scene> with Lys189. This combined movement locks the active site residues into a proper orientation for lysis of a C-C bond within isocitrate. <ref name="sharma"> Sharma, V.; Sharma, S.; Hoener zu Bentrup, K.; McKinney, J.; Russell, D.; ''et. al''; Structure of isocitrate lyase, a persistence factor of ''Mycobacterium tuberculosis''. ''Nat. Struct. Biol.''. '''2000'''. ''7(8)'':663-668. </ref>
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| ===Regulation===
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| <scene name='69/694225/Isocitrate_lyase/4'>Isocitrate lyase</scene> competes with [http://en.wikipedia.org/wiki/Isocitrate_dehydrogenase isocitrate dehydrogenase], an enzyme found in the [http://en.wikipedia.org/wiki/Citric_acid_cycle citric cycle], for isocitrate processing. The favoritism of one enzyme over the other is controlled by the phosphorylation of isocitrate dehydrogenase. This enzyme has a much higher affinity for isocitrate as compared to isocitrate lyase. Phosphorylation of isocitrate dehydrogenase inactivates the enzyme and leades to increased isocitrate lyase activity. <ref name="cozzone"> Cozzone, A.; Regulation of acetate metabolism by protein phosphorylation in enteric bacteria. ''Annual Review of Microbiology''. '''1998''', ''52'':127-164. doi: 10.1146/annurev.micro.52.1.127. </ref>
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| ==Mechanism of Action==
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| [[Image:Complete_Mechanism.PNG|500 px|right|thumb|'''Figure 5. Observed Mechanism for the Breakdown of Isocitrate by Isocitrate Lyase.''']]
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| Within <scene name='69/694225/Pka_shift/2'>isocitrate lyase</scene>, His193 shifts the pKa of Cys191 and removes its proton. This allows Cys191 to extract a proton from the hydroxyl group of isocitrate. The resulting oxyanion forms a carbonyl and forces the lysis of a C-C bond. Glyoxylate and the enol form of succinate are formed and stabilized with a Mg<sup>2+</sup> ion. The succinate enolate resonates and extracts the proton back from Cys191 to form succinate ('''Figure 5''').
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| ==Disease Association==
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| ===Clinical Implications===
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| [[Image:TCA.png|500 px|left|thumb|'''Figure 6. Citric Acid Cycle with Glyoxylate Shunt Pathway.''' In several bacterial species, there is a carbon conserving gloxylate shunt pathway that converts isocitrate to malate in two steps instead of the usual five steps.]] ''Mycobacterium tuberculosis'' is a respiratory infection that causes numerous fatalities throughout the world. It lives in organisms and feeds off of host cells, which indicate a variety of lipases exist within ''M. tuberculosis''. Current drugs that are on the market now target a small number of bacterial processes like cell wall formation and chromosomal replication. Although several antibiotics exist, all of them target these same mechanisms of inhibition. These commonalities have led to the prevalence of different multi-drug resistant (MDR) tuberculosis strains. Due to the high level of resistance, finding a lasting treatment for MDR TB infections has become very problematic. Studies into new mechanisms of inhibition will be crucial to prevent widespread outbreaks.
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| <scene name='69/694225/Isocitrate_lyase/4'>Isocitrate lyase</scene> plays a key role in survival of ''M. tuberculosis'' by sustaining intracellular infections in inflammatory respiratory macrophages.<ref name="muñoz-elías"> Muñoz-Elías, E.; McKinney, J.; ''M. tuberculosis'' isocitrate lyases 1 and 2 are jointly required for ''in vivo'' growth and virulence. ''Nat. Med.'' '''2005'''. ''11(6)'':638-644. doi:10.1038/nm1252. </ref> Used in the citric acid cycle, isocitrate lyase is the first enzyme catalyzing the carbon conserving glyoxylate pathway ('''Figure 6'''). This glyoxylate pathway has not been observed in mammals and thus presents a unique drug target to solely attack TB infections. Research has shown that upregulation of the glyoxylate cycle occurs for pathogens like ''M. tuberculosis'' during an infection. <ref name="srivastava"> Srivastava, V.; Janin, A.; Srivastava, B.; Srivastava, R.; Selection of genes of ''Mycobacterium tuberculosis'' upregulated during residence in lungs of infected mice. ''ScienceDirect''. '''2007'''. doi:10.1016/j.tube.2007.10.002. </ref>
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| ===Inhibitors===
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| Due to the increased usefulness of this enzyme in propagating ''M. tuberculosis'' infections, specific inhibitors are being looked into as possible therapeutic targets for isocitrate lyase. Two such inhibitors that have already been identified are [http://en.wikipedia.org/wiki/Bromopyruvic_acid bromopyruvate] and [http://en.wikipedia.org/wiki/Beta-Nitropropionic_acid nitropropionate]. Unfortunately, these molecules are non-specific and would also inhibit other enzymes essential for host function. <ref name="dunn"> Dunn, M.; Ramírez-Trujillo, J.; Hernández-Lucas, I.; Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. ''Microbiology''. '''2009'''. ''155'':3166-3175. doi:10.1099/mic.0.030858-0. </ref> More research is needed to identify inhibitors that selectively target enzymes in the glyoxylate cycle.
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| ==Other 3D Structures of Isocitrate Lyase==
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| *[http://www.rcsb.org/pdb/explore/explore.do?structureId=1F61 1F61] ''Mycobacterium tuberculosis''
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| *[http://www.rcsb.org/pdb/explore/explore.do?structureId=1F8M 1F8M] ''Mycobacterium tuberculosis''
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| *[http://www.rcsb.org/pdb/explore/explore.do?structureId=1DQU 1DQU] ''Aspergillus nidulans''
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| *[http://www.rcsb.org/pdb/explore/explore.do?structureId=1IGW 1IGW] ''Escherichia coli''
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| *[http://www.rcsb.org/pdb/explore/explore.do?structureId=3IG3 3IG3] ''Yersinia pestis''
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| *[http://www.rcsb.org/pdb/explore/explore.do?structureId=3I4E 3I4E] ''Burkholderia pseudomallei''
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| *[http://www.rcsb.org/pdb/explore/explore.do?structureId=3p0X 3P0X], [http://www.rcsb.org/pdb/explore/explore.do?structureId=3EOL 3EOL], [http://www.rcsb.org/pdb/explore/explore.do?structureId=3OQ8 3OQ8], [http://www.rcsb.org/pdb/explore/explore.do?structureId=3E5B 2E5B] ''Brucella melitensis''
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| </StructureSection> | | </StructureSection> |
| == References == | | == References == |
| <references/> | | <references/> |