4YCA

Evidence of Kinetic Cooperativity in dimeric Ketopantoate Reductase from Staphylococcus aureus

4YCA の概要

• エントリーDOI: 10.2210/pdb4yca/pdb
• 関連するPDBエントリー: 4S3M
• 分子名称: 2-dehydropantoate 2-reductase, NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE (3 entities in total)
• 機能のキーワード: reductase, dimer, rossmann, kinetic cooperativity, oxidoreductase
• 由来する生物種: Staphylococcus aureus
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 68362.72

構造登録者

Sanchez, J.E.,Gross, P.G.,Goetze, R.,Walsh Jr., R.M.,Peeples, W.B.,Wood, Z.A. (登録日: 2015-02-19, 公開日: 2015-03-11, 最終更新日: 2023-09-27)

主引用文献

Sanchez, J.E.,Gross, P.G.,Goetze, R.W.,Walsh Jr., R.M.,Peeples, W.B.,Wood, Z.A.
Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from Staphylococcus aureus.
Biochemistry, 54:3360-3369, 2015

PubMed Abstract: Ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A. Previous structural studies have been limited to Escherichia coli KPR, a monomeric enzyme that follows a sequential ordered mechanism. Here we report the crystal structure of the Staphylococcus aureus enzyme at 1.8 Å resolution, the first description of a dimeric KPR. Using sedimentation velocity analysis, we show that the S. aureus KPR dimer is stable in solution. In fact, our structural analysis shows that the dimeric assembly we identify is present in the majority of KPR crystal structures. Steady state analysis of S. aureus KPR reveals strong positive cooperativity with respect to NADPH (Hill coefficient of 2.5). In contrast, high concentrations of the substrate ketopantoate (KP) inhibit the activity of the enzyme. These observations are consistent with a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path. In fact, Förster resonance energy transfer studies of the equilibrium binding of NADPH show only a small degree of cooperativity between subunits (Hill coefficient of 1.3). Thus, the apparently strong cooperativity observed in substrate saturation curves is due to a kinetic process that favors NADPH binding first. This interpretation is consistent with our analysis of the A181L substitution, which increases the Km of ketopantoate 844-fold, without affecting kcat. The crystal structure of KPRA181L shows that the substitution displaces Ser239, which is known to be important for the binding affinity of KP. The decrease in KP affinity would enhance the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity. Consistent with this interpretation, the NADPH saturation curve for KPRA181L is hyperbolic.

PubMed: 25946571
DOI: 10.1021/acs.biochem.5b00174

実験手法

X-RAY DIFFRACTION (1.81 Å)