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| - | [[Image:2ggh.gif|left|200px]]<br /><applet load="2ggh" size="450" color="white" frame="true" align="right" spinBox="true" | |
| - | caption="2ggh, resolution 2.20Å" /> | |
| - | '''The mutant A68C-D72C-NLQ of Deinococcus Radiodurans Nacylamino acid racemase'''<br /> | |
| | | | |
| - | ==Overview== | + | ==The mutant A68C-D72C-NLQ of Deinococcus Radiodurans Nacylamino acid racemase== |
| - | N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid, amidohydrolase (D-NCAase) are important biocatalysts for producing, enantiopure alpha-amino acids. NAAAR forms an octameric assembly and, displays induced fit movements upon substrate binding, while D-NCAase is a, tetramer that does not change conformation in the presence of a ligand. To, investigate the effects of introducing potentially stabilizing S-S bridges, in these different multimeric enzymes, cysteine residues predicted to form, inter or intra-subunit disulfide bonds were introduced by site-directed, mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants, (A68C-D72C and P60C-Y100C) and two d-NCAase variants (A302C and, P295C-F304C). Intra-subunit S-S bonds were formed in two additional NAAAR, variants (E149C-A182C and V265C). Crystal structures of NAAARs variants, show limited deviations from the wild-type overall tertiary structure. An, apo A68C-D72C subunit differs from the wild-type enzyme, in which it has, an ordered lid loop, resembling ligand-bound NAAAR. The structures of, A222C and A302C D-NCAases are nearly identical to the wild-type enzyme., All mutants with inter-subunit bridges had increases in thermostability., Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar kcat/Km, ratios, whereas mutant D-NCAases demonstrated increased kcat/Km ratios at, high temperatures (A302C: 4.2-fold at 65 degrees C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the, fine-tuned catalytic site as temperature increases, achieving enhanced, activity. | + | <StructureSection load='2ggh' size='340' side='right'caption='[[2ggh]], [[Resolution|resolution]] 2.20Å' scene=''> |
| | + | == Structural highlights == |
| | + | <table><tr><td colspan='2'>[[2ggh]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Deinococcus_radiodurans Deinococcus radiodurans]. This structure supersedes the now removed PDB entries [http://oca.weizmann.ac.il/oca-bin/send-pdb?obs=1&id=2fkr 2fkr] and [http://oca.weizmann.ac.il/oca-bin/send-pdb?obs=1&id=2ba8 2ba8]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GGH OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2GGH FirstGlance]. <br> |
| | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.2Å</td></tr> |
| | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=NLQ:N~2~-ACETYL-L-GLUTAMINE'>NLQ</scene></td></tr> |
| | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=2ggh FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2ggh OCA], [https://pdbe.org/2ggh PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2ggh RCSB], [https://www.ebi.ac.uk/pdbsum/2ggh PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2ggh ProSAT]</span></td></tr> |
| | + | </table> |
| | + | == Function == |
| | + | [https://www.uniprot.org/uniprot/NSAR_DEIRA NSAR_DEIRA] Acts as a N-succinylamino acid racemase (NSAR) that catalyzes the racemization of N-succinyl-L-phenylglycine and N-succinyl-D/L-phenylalanine (PubMed:24872444, PubMed:25875730). Can catalyze the racemization of a broad range of N-acylamino acids, including N-acetyl-D/L-methionine, N-acetyl-D/L-phenylalanine, N-acetyl-L-glutamine, N-acetyl-L-tryptophan, N-acetyl-L-leucine, N-formyl-D-methionine, N-formyl-D-norleucine, N-carbamoyl-D-methionine and N-carbamoyl-D-norleucine (PubMed:15313614, PubMed:16650857, PubMed:25875730). Also converts 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) to 2-succinylbenzoate (OSB) (PubMed:24872444). Catalyzes both N-succinylamino acid racemization and OSB synthesis at equivalent rates (PubMed:24872444). However, NSAR activity is probably the protein's biological function, because menaquinone biosynthesis genes are missing in this species (Probable).<ref>PMID:15313614</ref> <ref>PMID:16650857</ref> <ref>PMID:24872444</ref> <ref>PMID:25875730</ref> <ref>PMID:16740275</ref> |
| | + | == Evolutionary Conservation == |
| | + | [[Image:Consurf_key_small.gif|200px|right]] |
| | + | Check<jmol> |
| | + | <jmolCheckbox> |
| | + | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/gg/2ggh_consurf.spt"</scriptWhenChecked> |
| | + | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> |
| | + | <text>to colour the structure by Evolutionary Conservation</text> |
| | + | </jmolCheckbox> |
| | + | </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=2ggh ConSurf]. |
| | + | <div style="clear:both"></div> |
| | + | <div style="background-color:#fffaf0;"> |
| | + | == Publication Abstract from PubMed == |
| | + | N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) are important biocatalysts for producing enantiopure alpha-amino acids. NAAAR forms an octameric assembly and displays induced fit movements upon substrate binding, while D-NCAase is a tetramer that does not change conformation in the presence of a ligand. To investigate the effects of introducing potentially stabilizing S-S bridges in these different multimeric enzymes, cysteine residues predicted to form inter or intra-subunit disulfide bonds were introduced by site-directed mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants (A68C-D72C and P60C-Y100C) and two d-NCAase variants (A302C and P295C-F304C). Intra-subunit S-S bonds were formed in two additional NAAAR variants (E149C-A182C and V265C). Crystal structures of NAAARs variants show limited deviations from the wild-type overall tertiary structure. An apo A68C-D72C subunit differs from the wild-type enzyme, in which it has an ordered lid loop, resembling ligand-bound NAAAR. The structures of A222C and A302C D-NCAases are nearly identical to the wild-type enzyme. All mutants with inter-subunit bridges had increases in thermostability. Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar kcat/Km ratios, whereas mutant D-NCAases demonstrated increased kcat/Km ratios at high temperatures (A302C: 4.2-fold at 65 degrees C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity. |
| | | | |
| - | ==About this Structure==
| + | Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase.,Chiu WC, You JY, Liu JS, Hsu SK, Hsu WH, Shih CH, Hwang JK, Wang WC J Mol Biol. 2006 Jun 9;359(3):741-53. Epub 2006 Apr 18. PMID:16650857<ref>PMID:16650857</ref> |
| - | 2GGH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Deinococcus_radiodurans Deinococcus radiodurans] with MG and NLQ as [http://en.wikipedia.org/wiki/ligands ligands]. This structure superseeds the now removed PDB entries 2FKR and 2BA8. Active as [http://en.wikipedia.org/wiki/Amino-acid_racemase Amino-acid racemase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.1.1.10 5.1.1.10] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2GGH OCA].
| + | |
| | | | |
| - | ==Reference==
| + | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
| - | Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase., Chiu WC, You JY, Liu JS, Hsu SK, Hsu WH, Shih CH, Hwang JK, Wang WC, J Mol Biol. 2006 Jun 9;359(3):741-53. Epub 2006 Apr 18. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=16650857 16650857]
| + | </div> |
| - | [[Category: Amino-acid racemase]]
| + | <div class="pdbe-citations 2ggh" style="background-color:#fffaf0;"></div> |
| | + | == References == |
| | + | <references/> |
| | + | __TOC__ |
| | + | </StructureSection> |
| | [[Category: Deinococcus radiodurans]] | | [[Category: Deinococcus radiodurans]] |
| - | [[Category: Single protein]] | + | [[Category: Large Structures]] |
| - | [[Category: Chiu, W.C.]] | + | [[Category: Chiu WC]] |
| - | [[Category: Wang, W.C.]] | + | [[Category: Wang WC]] |
| - | [[Category: MG]]
| + | |
| - | [[Category: NLQ]]
| + | |
| - | [[Category: deinococcus radiodurans]]
| + | |
| - | [[Category: n-acylamino acid racemase]]
| + | |
| - | | + | |
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Sun Nov 25 08:53:53 2007''
| + | |
| Structural highlights
Function
NSAR_DEIRA Acts as a N-succinylamino acid racemase (NSAR) that catalyzes the racemization of N-succinyl-L-phenylglycine and N-succinyl-D/L-phenylalanine (PubMed:24872444, PubMed:25875730). Can catalyze the racemization of a broad range of N-acylamino acids, including N-acetyl-D/L-methionine, N-acetyl-D/L-phenylalanine, N-acetyl-L-glutamine, N-acetyl-L-tryptophan, N-acetyl-L-leucine, N-formyl-D-methionine, N-formyl-D-norleucine, N-carbamoyl-D-methionine and N-carbamoyl-D-norleucine (PubMed:15313614, PubMed:16650857, PubMed:25875730). Also converts 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) to 2-succinylbenzoate (OSB) (PubMed:24872444). Catalyzes both N-succinylamino acid racemization and OSB synthesis at equivalent rates (PubMed:24872444). However, NSAR activity is probably the protein's biological function, because menaquinone biosynthesis genes are missing in this species (Probable).[1] [2] [3] [4] [5]
Evolutionary Conservation
Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.
Publication Abstract from PubMed
N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) are important biocatalysts for producing enantiopure alpha-amino acids. NAAAR forms an octameric assembly and displays induced fit movements upon substrate binding, while D-NCAase is a tetramer that does not change conformation in the presence of a ligand. To investigate the effects of introducing potentially stabilizing S-S bridges in these different multimeric enzymes, cysteine residues predicted to form inter or intra-subunit disulfide bonds were introduced by site-directed mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants (A68C-D72C and P60C-Y100C) and two d-NCAase variants (A302C and P295C-F304C). Intra-subunit S-S bonds were formed in two additional NAAAR variants (E149C-A182C and V265C). Crystal structures of NAAARs variants show limited deviations from the wild-type overall tertiary structure. An apo A68C-D72C subunit differs from the wild-type enzyme, in which it has an ordered lid loop, resembling ligand-bound NAAAR. The structures of A222C and A302C D-NCAases are nearly identical to the wild-type enzyme. All mutants with inter-subunit bridges had increases in thermostability. Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar kcat/Km ratios, whereas mutant D-NCAases demonstrated increased kcat/Km ratios at high temperatures (A302C: 4.2-fold at 65 degrees C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity.
Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase.,Chiu WC, You JY, Liu JS, Hsu SK, Hsu WH, Shih CH, Hwang JK, Wang WC J Mol Biol. 2006 Jun 9;359(3):741-53. Epub 2006 Apr 18. PMID:16650857[6]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Wang WC, Chiu WC, Hsu SK, Wu CL, Chen CY, Liu JS, Hsu WH. Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans. J Mol Biol. 2004 Sep 3;342(1):155-69. PMID:15313614 doi:http://dx.doi.org/10.1016/j.jmb.2004.07.023
- ↑ Chiu WC, You JY, Liu JS, Hsu SK, Hsu WH, Shih CH, Hwang JK, Wang WC. Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase. J Mol Biol. 2006 Jun 9;359(3):741-53. Epub 2006 Apr 18. PMID:16650857 doi:http://dx.doi.org/10.1016/j.jmb.2006.03.063
- ↑ Odokonyero D, Sakai A, Patskovsky Y, Malashkevich VN, Fedorov AA, Bonanno JB, Fedorov EV, Toro R, Agarwal R, Wang C, Ozerova ND, Yew WS, Sauder JM, Swaminathan S, Burley SK, Almo SC, Glasner ME. Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family. Proc Natl Acad Sci U S A. 2014 May 28. pii: 201318703. PMID:24872444 doi:http://dx.doi.org/10.1073/pnas.1318703111
- ↑ Soriano-Maldonado P, Andújar-Sánchez M, Clemente-Jiménez JM, Rodríguez-Vico F, Las Heras-Vázquez FJ, Martínez-Rodríguez S. Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49. Mol Biotechnol. 2015 May;57(5):454-65. PMID:25875730 doi:10.1007/s12033-015-9839-4
- ↑ Glasner ME, Fayazmanesh N, Chiang RA, Sakai A, Jacobson MP, Gerlt JA, Babbitt PC. Evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase family of the enolase superfamily. J Mol Biol. 2006 Jun 30;360(1):228-50. PMID:16740275 doi:10.1016/j.jmb.2006.04.055
- ↑ Chiu WC, You JY, Liu JS, Hsu SK, Hsu WH, Shih CH, Hwang JK, Wang WC. Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase. J Mol Biol. 2006 Jun 9;359(3):741-53. Epub 2006 Apr 18. PMID:16650857 doi:http://dx.doi.org/10.1016/j.jmb.2006.03.063
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