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| - | [[Image:2hyp.gif|left|200px]]<br /><applet load="2hyp" size="350" color="white" frame="true" align="right" spinBox="true" | |
| - | caption="2hyp, resolution 2.050Å" /> | |
| - | '''Crystal structure of Rv0805 D66A mutant'''<br /> | |
| | | | |
| - | ==Overview== | + | ==Crystal structure of Rv0805 D66A mutant== |
| - | Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.
| + | <StructureSection load='2hyp' size='340' side='right'caption='[[2hyp]], [[Resolution|resolution]] 2.05Å' scene=''> |
| - | | + | == Structural highlights == |
| - | ==About this Structure== | + | <table><tr><td colspan='2'>[[2hyp]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Mycobacterium_tuberculosis_H37Rv Mycobacterium tuberculosis H37Rv]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2HYP OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2HYP FirstGlance]. <br> |
| - | 2HYP is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis] with <scene name='pdbligand=FE:'>FE</scene>, <scene name='pdbligand=MN:'>MN</scene>, <scene name='pdbligand=BR:'>BR</scene> and <scene name='pdbligand=CAC:'>CAC</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/3',5'-cyclic-nucleotide_phosphodiesterase 3',5'-cyclic-nucleotide phosphodiesterase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.4.17 3.1.4.17] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2HYP OCA].
| + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.05Å</td></tr> |
| - | | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=BR:BROMIDE+ION'>BR</scene>, <scene name='pdbligand=CAC:CACODYLATE+ION'>CAC</scene>, <scene name='pdbligand=FE:FE+(III)+ION'>FE</scene>, <scene name='pdbligand=MN:MANGANESE+(II)+ION'>MN</scene></td></tr> |
| - | ==Reference== | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=2hyp FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2hyp OCA], [https://pdbe.org/2hyp PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2hyp RCSB], [https://www.ebi.ac.uk/pdbsum/2hyp PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2hyp ProSAT]</span></td></tr> |
| - | Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis., Shenoy AR, Capuder M, Draskovic P, Lamba D, Visweswariah SS, Podobnik M, J Mol Biol. 2007 Jan 5;365(1):211-25. Epub 2006 Oct 6. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17059828 17059828]
| + | </table> |
| - | [[Category: 3',5'-cyclic-nucleotide phosphodiesterase]] | + | == Function == |
| - | [[Category: Mycobacterium tuberculosis]]
| + | [https://www.uniprot.org/uniprot/CNPD3_MYCTU CNPD3_MYCTU] Cyclic nucleotide phosphodiesterase with a dual-specificity for the second messengers cAMP and cGMP (PubMed:16313172, PubMed:18757371). Can use 2',3'-cAMP, 2',3'-cGMP, 3',5'-cAMP, 3',5'-cGMP and 3',5'-cUMP (PubMed:16313172, PubMed:18757371, PubMed:19801656). Hydrolysis of 2',3'-cAMP produces a mixture of 3'-AMP (major product) and 2'-AMP (minor product) (PubMed:18757371). In vitro, is 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP (PubMed:18757371). Can also hydrolyze the model substrates p-nitrophenyl phosphate (pNPP), bis-(p-nitrophenyl phosphate) (bis(pNPP)) and p-nitrophenyl phenylphosphonate (pNPPP) (PubMed:16313172, PubMed:18757371, PubMed:19801656). Plays an important regulatory role in modulating the intracellular concentration of cAMP, thereby influencing cAMP-dependent processes (PubMed:16313172). May play a role in pathogenicity, not only by hydrolyzing cAMP, but also by altering properties of the cell wall (PubMed:19801656).<ref>PMID:16313172</ref> <ref>PMID:18757371</ref> <ref>PMID:19801656</ref> Overexpression elicits a transcriptional response that is independent of the phosphodiesterase activity. It does not alter the levels of cAMP-CRP regulated genes, even though cAMP levels are reduced in cells.<ref>PMID:23835087</ref> |
| - | [[Category: Single protein]]
| + | == Evolutionary Conservation == |
| - | [[Category: Capuder, M.]]
| + | [[Image:Consurf_key_small.gif|200px|right]] |
| - | [[Category: Draskovic, P.]]
| + | Check<jmol> |
| - | [[Category: Lamba, D.]] | + | <jmolCheckbox> |
| - | [[Category: Podobnik, M.]] | + | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/hy/2hyp_consurf.spt"</scriptWhenChecked> |
| - | [[Category: Shenoy, A R.]]
| + | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> |
| - | [[Category: Visweswariah, S S.]]
| + | <text>to colour the structure by Evolutionary Conservation</text> |
| - | [[Category: BR]] | + | </jmolCheckbox> |
| - | [[Category: CAC]] | + | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=2hyp ConSurf]. |
| - | [[Category: FE]] | + | <div style="clear:both"></div> |
| - | [[Category: MN]] | + | == References == |
| - | [[Category: bi-nuclear active site]] | + | <references/> |
| - | [[Category: camp]] | + | __TOC__ |
| - | [[Category: metallophosphoesterase]] | + | </StructureSection> |
| - | [[Category: phosphodiesterase]] | + | [[Category: Large Structures]] |
| - | | + | [[Category: Mycobacterium tuberculosis H37Rv]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:47:12 2008''
| + | [[Category: Capuder M]] |
| | + | [[Category: Draskovic P]] |
| | + | [[Category: Lamba D]] |
| | + | [[Category: Podobnik M]] |
| | + | [[Category: Shenoy AR]] |
| | + | [[Category: Visweswariah SS]] |
| Structural highlights
Function
CNPD3_MYCTU Cyclic nucleotide phosphodiesterase with a dual-specificity for the second messengers cAMP and cGMP (PubMed:16313172, PubMed:18757371). Can use 2',3'-cAMP, 2',3'-cGMP, 3',5'-cAMP, 3',5'-cGMP and 3',5'-cUMP (PubMed:16313172, PubMed:18757371, PubMed:19801656). Hydrolysis of 2',3'-cAMP produces a mixture of 3'-AMP (major product) and 2'-AMP (minor product) (PubMed:18757371). In vitro, is 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP (PubMed:18757371). Can also hydrolyze the model substrates p-nitrophenyl phosphate (pNPP), bis-(p-nitrophenyl phosphate) (bis(pNPP)) and p-nitrophenyl phenylphosphonate (pNPPP) (PubMed:16313172, PubMed:18757371, PubMed:19801656). Plays an important regulatory role in modulating the intracellular concentration of cAMP, thereby influencing cAMP-dependent processes (PubMed:16313172). May play a role in pathogenicity, not only by hydrolyzing cAMP, but also by altering properties of the cell wall (PubMed:19801656).[1] [2] [3] Overexpression elicits a transcriptional response that is independent of the phosphodiesterase activity. It does not alter the levels of cAMP-CRP regulated genes, even though cAMP levels are reduced in cells.[4]
Evolutionary Conservation
Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.
References
- ↑ Shenoy AR, Sreenath N, Podobnik M, Kovacevic M, Visweswariah SS. The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis. Biochemistry. 2005 Dec 6;44(48):15695-704. PMID:16313172 doi:10.1021/bi0512391
- ↑ Keppetipola N, Shuman S. A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. J Biol Chem. 2008 Nov 7;283(45):30942-9. PMID:18757371 doi:10.1074/jbc.M805064200
- ↑ Podobnik M, Tyagi R, Matange N, Dermol U, Gupta AK, Mattoo R, Seshadri K, Visweswariah SS. A mycobacterial cyclic AMP phosphodiesterase that moonlights as a modifier of cell wall permeability. J Biol Chem. 2009 Nov 20;284(47):32846-57. Epub 2009 Sep 29. PMID:19801656 doi:10.1074/jbc.M109.049635
- ↑ Matange N, Hunt DM, Buxton RS, Visweswariah SS. Overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response. Tuberculosis (Edinb). 2013 Sep;93(5):492-500. PMID:23835087 doi:10.1016/j.tube.2013.05.004
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