The Crystal Structure of Escherichia coli Ketopantoate Reductase with NADP+ Bound
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- 作者：Carina M. C. Lobley ; Alessio Ciulli ; Heather M. Whitney ; Glyn Williams ; Alison G. Smith ; Chris Abell ; and Tom L. Blundell
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：June 28, 2005
- 年：2005
- 卷：44
- 期：25
- 页码：8930 - 8939
- 全文大小：632K
- 年卷期：v.44,no.25(June 28, 2005)
- ISSN：1520-4995
文摘
The NADPH-dependent reduction of ketopantoate to pantoate, catalyzed by ketopantoatereductase (KPR; EC 1.1.1.169), is essential for the biosynthesis of pantothenate (vitamin B5). Here wepresent the crystal structure of Escherichia coli KPR with NADP+ bound, solved to 2.1 Å resolution. Thecofactor is bound in the active site cleft between the N-terminal Rossmann-fold domain and the C-terminal
-helical domain. The thermodynamics of cofactor and substrate binding were characterized by isothermaltitration calorimetry. The dissociation constant for NADP+ was found to be 6.5 
M, 20-fold larger thanthat for NADPH (0.34 M). The difference is primarily due to the entropic term, suggesting favorablehydrophobic interactions of the more lipophilic nicotinamide ring in NADPH. Comparison of this binarycomplex structure with the previously studied apoenzyme reveals no evidence for large domain movementson cofactor binding. This observation is further supported both by molecular dynamics and by calorimetricanalysis. A model of the ternary complex, based on the structure presented here, provides novel insightsinto the molecular mechanism of enzyme catalysis. We propose a conformational switch of the essentialLys176 from the "resting" state observed in our structure to an "active" state, to bind ketopantoate.Additionally, we identify the importance of Asn98 for substrate binding and enzyme catalysis.
-helical domain. The thermodynamics of cofactor and substrate binding were characterized by isothermaltitration calorimetry. The dissociation constant for NADP+ was found to be 6.5 M, 20-fold larger thanthat for NADPH (0.34 M). The difference is primarily due to the entropic term, suggesting favorablehydrophobic interactions of the more lipophilic nicotinamide ring in NADPH. Comparison of this binarycomplex structure with the previously studied apoenzyme reveals no evidence for large domain movementson cofactor binding. This observation is further supported both by molecular dynamics and by calorimetricanalysis. A model of the ternary complex, based on the structure presented here, provides novel insightsinto the molecular mechanism of enzyme catalysis. We propose a conformational switch of the essentialLys176 from the "resting" state observed in our structure to an "active" state, to bind ketopantoate.Additionally, we identify the importance of Asn98 for substrate binding and enzyme catalysis.