Publications

• The crystal structure of Escherichia coli ketopantoate reductase with NADP+ bound.

  Lobley C.M.C., Ciulli A., Whitney H.M., Williams G., Smith A.G., Abell C., Blundell T.L.

  The NADPH-dependent reduction of ketopantoate to pantoate, catalyzed by ketopantoate reductase (KPR; EC 1.1.1.169), is essential for the biosynthesis of pantothenate (vitamin B(5)). Here we present the crystal structure of Escherichia coli KPR with NADP(+) bound, solved to 2.1 A resolution. The co ... >> More

  The NADPH-dependent reduction of ketopantoate to pantoate, catalyzed by ketopantoate reductase (KPR; EC 1.1.1.169), is essential for the biosynthesis of pantothenate (vitamin B(5)). Here we present the crystal structure of Escherichia coli KPR with NADP(+) bound, solved to 2.1 A resolution. The cofactor is bound in the active site cleft between the N-terminal Rossmann-fold domain and the C-terminal alpha-helical domain. The thermodynamics of cofactor and substrate binding were characterized by isothermal titration calorimetry. The dissociation constant for NADP(+) was found to be 6.5 muM, 20-fold larger than that for NADPH (0.34 muM). The difference is primarily due to the entropic term, suggesting favorable hydrophobic interactions of the more lipophilic nicotinamide ring in NADPH. Comparison of this binary complex structure with the previously studied apoenzyme reveals no evidence for large domain movements on cofactor binding. This observation is further supported both by molecular dynamics and by calorimetric analysis. A model of the ternary complex, based on the structure presented here, provides novel insights into the molecular mechanism of enzyme catalysis. We propose a conformational switch of the essential Lys176 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. << Less

  Biochemistry 44:8930-8939(2005) [PubMed] [EuropePMC]

• Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase.

  Zheng R., Blanchard J.S.

  Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the panE gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exis ... >> More

  Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the panE gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da. The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP(+) release. The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism. An enzyme group exhibiting a pK value of 8.4 +/-0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 +/-0.2 serves as a general base in the direction of pantoate oxidation. The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by (1)H NMR spectroscopy. Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion. The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, DeltaG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation. << Less

  Biochemistry 39:3708-3717(2000) [PubMed] [EuropePMC]

• 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 D.Y., Smith A.G., Blundell T.L., Abell C.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 282:8487-8497(2007) [PubMed] [EuropePMC]

• The panE gene, encoding ketopantoate reductase, maps at 10 minutes and is allelic to apbA in Salmonella typhimurium.

  Frodyma M.E., Downs D.M.

  In Salmonella typhimurium, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. The apbA gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. ... >> More

  In Salmonella typhimurium, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. The apbA gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. Petersen, J. Bacteriol. 176:4858-4864, 1994). Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid. This activity had previously been associated with the pantothenate biosynthetic gene panE. Although previous reports placed panE at 87 min on the Escherichia coli chromosome, we show herein that apbA and panE are allelic and map to 10 min on both the S. typhimurium and E. coli chromosomes. Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway. << Less

  J. Bacteriol. 180:4757-4759(1998) [PubMed] [EuropePMC]