1k41
From Proteopedia
(New page: 200px<br /><applet load="1k41" size="450" color="white" frame="true" align="right" spinBox="true" caption="1k41, resolution 2.2Å" /> '''Crystal structure of ...) |
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| - | [[Image:1k41.gif|left|200px]]<br /><applet load="1k41" size=" | + | [[Image:1k41.gif|left|200px]]<br /><applet load="1k41" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1k41, resolution 2.2Å" /> | caption="1k41, resolution 2.2Å" /> | ||
'''Crystal structure of KSI Y57S mutant'''<br /> | '''Crystal structure of KSI Y57S mutant'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a | + | Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices. |
==About this Structure== | ==About this Structure== | ||
| - | 1K41 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida]. Active as [http://en.wikipedia.org/wiki/Steroid_Delta-isomerase Steroid Delta-isomerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.3.3.1 5.3.3.1] Full crystallographic information is available from [http:// | + | 1K41 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida]. Active as [http://en.wikipedia.org/wiki/Steroid_Delta-isomerase Steroid Delta-isomerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.3.3.1 5.3.3.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1K41 OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Steroid Delta-isomerase]] | [[Category: Steroid Delta-isomerase]] | ||
| - | [[Category: Cha, S | + | [[Category: Cha, S S.]] |
| - | [[Category: Choi, K | + | [[Category: Choi, K Y.]] |
| - | [[Category: Jang, D | + | [[Category: Jang, D S.]] |
| - | [[Category: Lee, T | + | [[Category: Lee, T H.]] |
| - | [[Category: Nam, G | + | [[Category: Nam, G H.]] |
| - | [[Category: Oh, B | + | [[Category: Oh, B H.]] |
[[Category: ksi y57s helix]] | [[Category: ksi y57s helix]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:29:53 2008'' |
Revision as of 11:29, 21 February 2008
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Crystal structure of KSI Y57S mutant
Overview
Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.
About this Structure
1K41 is a Single protein structure of sequence from Pseudomonas putida. Active as Steroid Delta-isomerase, with EC number 5.3.3.1 Full crystallographic information is available from OCA.
Reference
Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B., Nam GH, Jang DS, Cha SS, Lee TH, Kim DH, Hong BH, Yun YS, Oh BH, Choi KY, Biochemistry. 2001 Nov 13;40(45):13529-37. PMID:11695900
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