2ggh
From Proteopedia
(New page: 200px<br /><applet load="2ggh" size="450" color="white" frame="true" align="right" spinBox="true" caption="2ggh, resolution 2.20Å" /> '''The mutant A68C-D72C...) |
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| - | [[Image:2ggh.gif|left|200px]]<br /><applet load="2ggh" size=" | + | [[Image:2ggh.gif|left|200px]]<br /><applet load="2ggh" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2ggh, resolution 2.20Å" /> | caption="2ggh, resolution 2.20Å" /> | ||
'''The mutant A68C-D72C-NLQ of Deinococcus Radiodurans Nacylamino acid racemase'''<br /> | '''The mutant A68C-D72C-NLQ of Deinococcus Radiodurans Nacylamino acid racemase'''<br /> | ||
==Overview== | ==Overview== | ||
| - | N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid | + | 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== | ==About this Structure== | ||
| - | 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 | + | 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 <scene name='pdbligand=MG:'>MG</scene> and <scene name='pdbligand=NLQ:'>NLQ</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. This structure supersedes 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://oca.weizmann.ac.il/oca-bin/ocashort?id=2GGH OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Deinococcus radiodurans]] | [[Category: Deinococcus radiodurans]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Chiu, W | + | [[Category: Chiu, W C.]] |
| - | [[Category: Wang, W | + | [[Category: Wang, W C.]] |
[[Category: MG]] | [[Category: MG]] | ||
[[Category: NLQ]] | [[Category: NLQ]] | ||
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[[Category: n-acylamino acid racemase]] | [[Category: n-acylamino acid racemase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:31:26 2008'' |
Revision as of 15:31, 21 February 2008
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The mutant A68C-D72C-NLQ of Deinococcus Radiodurans Nacylamino acid racemase
Overview
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
2GGH is a Single protein structure of sequence from Deinococcus radiodurans with and as ligands. This structure supersedes the now removed PDB entries 2FKR and 2BA8. Active as Amino-acid racemase, with EC number 5.1.1.10 Full crystallographic information is available from OCA.
Reference
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
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