A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E. coli Phosphofructokinase-2

Báez, María E.; Cabrera, Ricardo; Pereira, H.M.; Blanco, Alejandro; Villalobos, Pablo; Ramírez‐Sarmiento, César A.; Caniuguir, A.; Guixé, Victoria; Garratt, R.C.; Babul, Jorge

doi:10.1016/j.bpj.2013.05.028

Cited by 11 publications

(9 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although the estimated radius from SEC at protein concentrations of 35 mM is larger than the value obtained from DLS measurements at 30 mM ( Fig. 2 C), this could be due to the presence of potassium, which is known to bind, stabilize, and enhance the activity of WT Pfk-2 (36). At protein concentrations above 30 mM, the hydrodynamic radius of L93A Pfk-2 is considerably larger, being 3.4 5 0.2 nm at 120 mM according to DLS measurements (Fig.…”

Section: Resultsmentioning

confidence: 70%

The Folding Unit of Phosphofructokinase-2 as Defined by the Biophysical Properties of a Monomeric Mutant

Ramírez‐Sarmiento

Báez

Zamora

et al. 2015

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened β-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 μM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240-309. These residues correspond to most of the β-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the β-meander module is not affected by monomerizing mutations.

show abstract

Section: Resultsmentioning

confidence: 70%

The Folding Unit of Phosphofructokinase-2 as Defined by the Biophysical Properties of a Monomeric Mutant

Ramírez‐Sarmiento

Báez

Zamora

et al. 2015

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…It has been previously shown that mono- or divalent metal ions, such as K + , Zn 2+ , and Mg 2+ , are essential for catalysis in many kinases, where their concerted action is the driving force for ATP binding and substrate catalysis. − Accordingly, the crystal structure shows electron density in the vicinity of the AMP-PCP/ADP phosphate groups, which was modeled as a water molecule since no Mg 2+ was present in the crystallization conditions (Supporting Information Figure S8).…”

Section: Resultsmentioning

confidence: 99%

A Subfamily of Bacterial Ribokinases Utilizes a Hemithioacetal for Pyridoxal Phosphate Salvage

et al. 2014

View full text Add to dashboard Cite

Pyridoxal 5'-phosphate (PLP) is the active vitamer of vitamin B6 and acts as an essential cofactor in many aspects of amino acid and sugar metabolism. The virulence and survival of pathogenic bacteria such as Mycobacterium tuberculosis depend on PLP, and deficiencies in humans have also been associated with neurological disorders and inflammation. While PLP can be synthesized by a de novo pathway in bacteria and plants, most higher organisms rely on a salvage pathway that phosphorylates either pyridoxal (PL) or its related vitamers, pyridoxine (PN) and pyridoxamine (PM). PL kinases (PLKs) are essential for this phosphorylation step and are thus of major importance for cellular viability. We recently identified a pyridoxal kinase (SaPLK) as a target of the natural product antibiotic rugulactone (Ru) in Staphylococcus aureus. Surprisingly, Ru selectively modified SaPLK not at the active site cysteine, but on a remote cysteine residue. Based on structural and biochemical studies, we now provide insight into an unprecedented dual Cys charge relay network that is mandatory for PL phosphorylation. The key component is the reactive Cys 110 residue in the lid region that forms a hemithioactetal intermediate with the 4'-aldehyde of PL. This hemithioacetal, in concert with the catalytic Cys 214, increases the nucleophilicity of the PL 5'-OH group for the inline displacement reaction with the γ-phosphate of ATP. A closer inspection of related enzymes reveals that Cys 110 is conserved and thus serves as a characteristic mechanistic feature for a dual-function ribokinase subfamily herein termed CC-PLKs.

show abstract

“…The E. coli phosphofructokinase-2 shows elegant allosteric regulation by Mg-ATP (as ATP is both a substrate and the end product of glycolysis). A second Mg-ATP molecule binds close to the active site, slowing the reaction without competing with the carbohydrate substrate [71,72]. RKs are allosterically regulated by a range of phosphorylated products [73][74][75].…”

Section: Allosteric Regulation Of Ribokinasesmentioning

confidence: 99%

Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds

2019

View full text Add to dashboard Cite

Carbohydrate kinases activate a wide variety of monosaccharides by adding a phosphate group, usually from ATP. This modification is fundamental to saccharide utilization, and it is likely a very ancient reaction. Modern organisms contain carbohydrate kinases from at least five main protein families. These range from the highly specialized inositol kinases, to the ribokinases and galactokinases, which belong to families that phosphorylate a wide range of substrates. The carbohydrate kinases utilize a common strategy to drive the reaction between the sugar hydroxyl and the donor phosphate. Each sugar is held in position by a network of hydrogen bonds to the non-reactive hydroxyls (and other functional groups). The reactive hydroxyl is deprotonated, usually by an aspartic acid side chain acting as a catalytic base. The deprotonated hydroxyl then attacks the donor phosphate. The resulting pentacoordinate transition state is stabilized by an adjacent divalent cation, and sometimes by a positively charged protein side chain or the presence of an anion hole. Many carbohydrate kinases are allosterically regulated using a wide variety of strategies, due to their roles at critical control points in carbohydrate metabolism. The evolution of a similar mechanism in several folds highlights the elegance and simplicity of the catalytic scheme.

show abstract

A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E. coli Phosphofructokinase-2

Cited by 11 publications

References 44 publications

The Folding Unit of Phosphofructokinase-2 as Defined by the Biophysical Properties of a Monomeric Mutant

The Folding Unit of Phosphofructokinase-2 as Defined by the Biophysical Properties of a Monomeric Mutant

A Subfamily of Bacterial Ribokinases Utilizes a Hemithioacetal for Pyridoxal Phosphate Salvage

Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds

Contact Info

Product

Resources

About