1ocv: Difference between revisions

Revision as of 15:16, 21 February 2008

THE F116W MUTANT STRUCTURE OF KETOSTEROID ISOMERASE FROM COMAMONAS TESTOSTERONI

OverviewOverview

Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.

About this StructureAbout this Structure

1OCV is a Single protein structure of sequence from Comamonas testosteroni. Active as Steroid Delta-isomerase, with EC number 5.3.3.1 Full crystallographic information is available from OCA.

ReferenceReference

Origin of the different pH activity profile in two homologous ketosteroid isomerases., Yun YS, Lee TH, Nam GH, Jang DS, Shin S, Oh BH, Choi KY, J Biol Chem. 2003 Jul 25;278(30):28229-36. Epub 2003 May 6. PMID:12734184

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 '''THE F116W MUTANT STRUCTURE OF KETOSTEROID ISOMERASE FROM COMAMONAS TESTOSTERONI'''<br />
 ==Overview==
-Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni, (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH, activity profiles. TI-WT loses activity below pH 5.0 due to the, protonation of the conserved catalytic base, Asp-38, while PI-WT does not., Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of, Trp-116, which is homologously replaced with Phe-116 in TI-WT. To, investigate the role of Trp-116, we prepared the F116W mutant of TI-WT, (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic, parameters of those mutants at different pH levels. PI-W116F exhibited, significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and, increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH, 3.8. The crystal structure of TI-F116W clearly showed that the indole ring, NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of, Asp-38 like that of PI-WT. The present results demonstrate that the, activities of both PI-WT and TI-F116W at low pH were maintained by a, tryptophan, which was able not only to lower the pKa value of the, catalytic base but also to increase the substrate affinity. This is one, example of the strategy nature can adopt to evolve the diversity of the, catalytic function in the enzymes. Our results provide insight into, deciphering the molecular evolution of the enzyme and creating novel, enzymes by protein engineering.
+Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.
 ==About this Structure==
-OCV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Comamonas_testosteroni Comamonas testosteroni]. 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://ispc.weizmann.ac.il/oca-bin/ocashort?id=1OCV OCA].
+OCV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Comamonas_testosteroni Comamonas testosteroni]. 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=1OCV OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: Steroid Delta-isomerase]]
-[[Category: Lee, T.H.]]
+[[Category: Lee, T H.]]
 [[Category: Shin, S.]]
-[[Category: Yun, Y.S.]]
+[[Category: Yun, Y S.]]
 [[Category: delta-5-3-ketosteroid]]
 [[Category: ketosteroid isomerase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 22:55:13 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:16:11 2008''