1get: Difference between revisions
New page: left|200px<br /><applet load="1get" size="450" color="white" frame="true" align="right" spinBox="true" caption="1get, resolution 2.00Å" /> '''ANATOMY OF AN ENGINE... |
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[[Image:1get.gif|left|200px]]<br /><applet load="1get" size=" | [[Image:1get.gif|left|200px]]<br /><applet load="1get" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1get, resolution 2.00Å" /> | caption="1get, resolution 2.00Å" /> | ||
'''ANATOMY OF AN ENGINEERED NAD-BINDING SITE'''<br /> | '''ANATOMY OF AN ENGINEERED NAD-BINDING SITE'''<br /> | ||
==Overview== | ==Overview== | ||
The coenzyme specificity of Escherichia coli glutathione reductase was | The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate. | ||
==About this Structure== | ==About this Structure== | ||
1GET is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with FAD and NAP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Glutathione-disulfide_reductase Glutathione-disulfide reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.8.1.7 1.8.1.7] Full crystallographic information is available from [http:// | 1GET is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=FAD:'>FAD</scene> and <scene name='pdbligand=NAP:'>NAP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Glutathione-disulfide_reductase Glutathione-disulfide reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.8.1.7 1.8.1.7] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1GET OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Glutathione-disulfide reductase]] | [[Category: Glutathione-disulfide reductase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Mittl, P | [[Category: Mittl, P R.E.]] | ||
[[Category: Schulz, G | [[Category: Schulz, G E.]] | ||
[[Category: FAD]] | [[Category: FAD]] | ||
[[Category: NAP]] | [[Category: NAP]] | ||
[[Category: oxidoreductase(flavoenzyme)]] | [[Category: oxidoreductase(flavoenzyme)]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:49:15 2008'' | ||
Revision as of 13:49, 21 February 2008
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ANATOMY OF AN ENGINEERED NAD-BINDING SITE
OverviewOverview
The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate.
About this StructureAbout this Structure
1GET is a Single protein structure of sequence from Escherichia coli with and as ligands. Active as Glutathione-disulfide reductase, with EC number 1.8.1.7 Full crystallographic information is available from OCA.
ReferenceReference
Anatomy of an engineered NAD-binding site., Mittl PR, Berry A, Scrutton NS, Perham RN, Schulz GE, Protein Sci. 1994 Sep;3(9):1504-14. PMID:7833810
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