5z2b: Difference between revisions
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==Crystal structure of highly active BTUO mutant P287G Improved by Humidity Control at 86% RH== | |||
<StructureSection load='5z2b' size='340' side='right' caption='[[5z2b]], [[Resolution|resolution]] 1.90Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[5z2b]] is a 2 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5Z2B OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5Z2B FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=AZA:8-AZAXANTHINE'>AZA</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=OXY:OXYGEN+MOLECULE'>OXY</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr> | |||
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4xfp|4xfp]], [[5y52|5y52]], [[5yja|5yja]]</td></tr> | |||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Factor_independent_urate_hydroxylase Factor independent urate hydroxylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.7.3.3 1.7.3.3] </span></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5z2b FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5z2b OCA], [http://pdbe.org/5z2b PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5z2b RCSB], [http://www.ebi.ac.uk/pdbsum/5z2b PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5z2b ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[[http://www.uniprot.org/uniprot/PUCL_BACSB PUCL_BACSB]] Catalyzes two steps in the degradation of uric acid, i.e. the oxidation of uric acid to 5-hydroxyisourate (HIU) and the stereoselective decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) to (S)-allantoin (By similarity). | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two beta-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by approximately 20 degrees C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 A resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cbeta distance between Arg298 residues of the wild-type enzyme (5.4 A apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins. | |||
Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity.,Hibi T, Kume A, Kawamura A, Itoh T, Fukada H, Nishiya Y Biochemistry. 2016 Jan 15. PMID:26739254<ref>PMID:26739254</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
<div class="pdbe-citations 5z2b" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Factor independent urate hydroxylase]] | |||
[[Category: Hibi, T]] | |||
[[Category: Itoh, T]] | |||
[[Category: Nishiya, Y]] | [[Category: Nishiya, Y]] | ||
[[Category: | [[Category: Entropy of activation]] | ||
[[Category: | [[Category: Enzyme]] | ||
[[Category: Loop flexibility]] | |||
[[Category: Oxidoreductase]] | |||
[[Category: Protein engineering]] | |||
Revision as of 09:06, 2 January 2019
Crystal structure of highly active BTUO mutant P287G Improved by Humidity Control at 86% RHCrystal structure of highly active BTUO mutant P287G Improved by Humidity Control at 86% RH
Structural highlights
Function[PUCL_BACSB] Catalyzes two steps in the degradation of uric acid, i.e. the oxidation of uric acid to 5-hydroxyisourate (HIU) and the stereoselective decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) to (S)-allantoin (By similarity). Publication Abstract from PubMedBacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two beta-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by approximately 20 degrees C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 A resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cbeta distance between Arg298 residues of the wild-type enzyme (5.4 A apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins. Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity.,Hibi T, Kume A, Kawamura A, Itoh T, Fukada H, Nishiya Y Biochemistry. 2016 Jan 15. PMID:26739254[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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