2ofp
From Proteopedia
(New page: 200px<br /><applet load="2ofp" size="450" color="white" frame="true" align="right" spinBox="true" caption="2ofp, resolution 2.30Å" /> '''Crystal structure of...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:2ofp.gif|left|200px]]<br /><applet load="2ofp" size=" | + | [[Image:2ofp.gif|left|200px]]<br /><applet load="2ofp" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2ofp, resolution 2.30Å" /> | caption="2ofp, resolution 2.30Å" /> | ||
'''Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate'''<br /> | '''Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent | + | Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B5). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP+ and pantoate bound, solved to 2.3 A of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn98 as well as Lys176 and Glu256 in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser244 for substrate binding and of Arg31 and Lys72 for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity. |
==About this Structure== | ==About this Structure== | ||
| - | 2OFP 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 ACT, NAP, PAF and DIO as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/2-dehydropantoate_2-reductase 2-dehydropantoate 2-reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.169 1.1.1.169] Full crystallographic information is available from [http:// | + | 2OFP 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=ACT:'>ACT</scene>, <scene name='pdbligand=NAP:'>NAP</scene>, <scene name='pdbligand=PAF:'>PAF</scene> and <scene name='pdbligand=DIO:'>DIO</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/2-dehydropantoate_2-reductase 2-dehydropantoate 2-reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.169 1.1.1.169] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2OFP OCA]. |
==Reference== | ==Reference== | ||
| Line 15: | Line 15: | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Abell, C.]] | [[Category: Abell, C.]] | ||
| - | [[Category: Blundell, T | + | [[Category: Blundell, T L.]] |
| - | [[Category: Chirgadze, D | + | [[Category: Chirgadze, D Y.]] |
[[Category: Ciulli, A.]] | [[Category: Ciulli, A.]] | ||
| - | [[Category: Smith, A | + | [[Category: Smith, A G.]] |
[[Category: ACT]] | [[Category: ACT]] | ||
[[Category: DIO]] | [[Category: DIO]] | ||
| Line 28: | Line 28: | ||
[[Category: ternary complex]] | [[Category: ternary complex]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:17:56 2008'' |
Revision as of 16:17, 21 February 2008
|
Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate
Overview
Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B5). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP+ and pantoate bound, solved to 2.3 A of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn98 as well as Lys176 and Glu256 in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser244 for substrate binding and of Arg31 and Lys72 for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity.
About this Structure
2OFP is a Single protein structure of sequence from Escherichia coli with , , and as ligands. Active as 2-dehydropantoate 2-reductase, with EC number 1.1.1.169 Full crystallographic information is available from OCA.
Reference
Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity., Ciulli A, Chirgadze DY, Smith AG, Blundell TL, Abell C, J Biol Chem. 2007 Mar 16;282(11):8487-97. Epub 2007 Jan 16. PMID:17229734
Page seeded by OCA on Thu Feb 21 18:17:56 2008
Categories: 2-dehydropantoate 2-reductase | Escherichia coli | Single protein | Abell, C. | Blundell, T L. | Chirgadze, D Y. | Ciulli, A. | Smith, A G. | ACT | DIO | NAP | PAF | Apba | Ketopantoate reductase | Pane | Ternary complex
